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Graduate School
1983
Equine Infectious Anemia Virus: Characterization
of Virion Polypeptides and the Biochemical Nature
of Antigenic Variation.
Bharat Savailal Parekh
Louisiana State University and Agricultural & Mechanical College
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Antigenic Variation." (1983). LSU Historical Dissertations and Theses. 3903.
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EQUINE INFECTIOUS ANEMIA VIRUS: CHARACTERIZATION OF VIRION
POLYPEPTIDES AND THE BIOCHEMICAL NATURE OF ANTIGENIC
VARIATION
The L ou isia n a State University and A g ric u ltu ra l a n d M ech an ical Col.
University
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EQUINE INFECTIOUS ANEMIA VIRUS: CHARACTERIZATION OF VIRION
POLYPEPTIDES AND THE BIOCHEMICAL NATURE OF ANTIGENIC VARIATION.
A dissertation
Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in
The Department of Biochemistry
by
Bharat S. Parekh
B.S., University of Bombay, 1973
M.S., University of Bombay, 1975
Ph.D., August, 1983
ACKNOWLEDGEMENTS
I would like to express my thanks and sincere appreciation to my
major professor Dr. R. C. Montelaro for his guidance, encouragement
and constant support during the period of this investigation and the
preparation of this dissertation.
Working with him has been a very
stimulating and rewarding experience.
I would also like to express my sincere thanks to Dr. C. J.
Issel, who has been always ready to help, discuss and provide all
assistance including tissue culture and dark room facilities.
I am also thankful to Dr. Alberto Orrego, Melanie West, Tammy
Allgood, Nancy Lohrey, Vera Barta and Helen Dworin for helping me from
time to time during the course of this research, and Janet Evans for
her expert help in the preparation of this dissertation.
ii
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENT ..............................................
ii
TABLE OF CONTENTS.............................................. iii
LIST OF T A B L E S ..............................................
LIST OF FIGURES . . ....................................
iv
v
A B S T R A C T ...................................................... vii
................................................
1
MATERIALS AND METHODS ........................................
33
RESULTS......................................................
52
INTRODUCTION
D I S C U S S I O N .................................................... Ill
LITERATURE CITED... ...........................................
126
APPENDIX I (LIST OF ABBREVIATIONS)............................. 139
V I T A .......................................................... 140
iii
LIST OF TABLES
Table
Page
..................
1-1
Taxonomic features of Retroviridae
1-2
Structural polypeptides of avian and murine type-C
retroviruses
......................................
6
12
III-l
Polypeptides of equine infectious anemia virus . . . .
III-2
lodination efficiencies of EIAV polypeptides
IV-1
Polypeptide composition of EIAV, FLV and A S V .......... 117
iv
........
66
80
LIST OF FIGURES
Figures
Page
1-1
A model of retroviruses.....................
10
1-2
Replication scheme of retrovirus genome ..............
16
1-3
Sequence of events in an animal suffering with chronic
E I A V ..............................................
31
II-l
Generation scheme of EIAV i s o l a t e s ..................
36
III-l
SDS-PAGE analysis of radioactively labeled EIAV . . . .
53
III-2
GHC1-GF analysis of radioactively labeled EIAV
....
57
III-3
SDS-PAGE analysis of the individual components
...........
60
obtained by GHC1-GF of [^H]-leucine EIAV
III-4
SDS-PAGE analysis of the individual components
obtained by GHC1-GF of [^H]-glucosamine EIAV
III-5
........
SDS-PAGE analysis of peptide fragments after limited
3
proteolytic digestion of [ H]-leucine polypeptides . .
125
III-6
Two-dimensional tryptic peptide analysis of
III-7
labeled EIAV proteins...............................
3
Isoelectric focussing of [ H]-leucine labeled EIAV
polypeptides
63
69
I
71
......................................
74
III-8
Phosphoprotein analysis .............................
76
III-9
SDS-PAGE analysis of bromelain treated radioactive
E I A V ..............................................
111-10
SDS-PAGE analysis of surface radioiodinated EIAV
III-l1
Ribonucleoprotein complex preparation and analysis
111-12
A tentative model of E I A V ...........................
v
...
. .
82
84
86
90
Figures
111-13
Page
Rectal temperature plots of experimentally inoculated
p o n i e s ............................................
92
111-14
SDS-PAGE analysis of proteins of EIAV isolates
94
111-15
SDS-PAGE analysis of glycoproteins of EIAV isolates . .
111-16
Two-dimensional peptide analysis of gp90 tryptic
....
digests from EIAV i s o l a t e s .........................
111-17
95
98
Two-dimensional peptide analysis of gp45 tryptic
digests from EIAV isolates
................... 101
111-18
Glycopeptide analysis ...............................
111-19
Two-dimensional peptide analysis of p26 tryptic
104
digests from EIAV i s o l a t e s ........................... 106
111-20
Two-dimensional peptide analysis of ppl5 tryptic
digests from EIAV i s o l a t e s ........................... 108
111-21
Two-dimensional peptide analysis of p9 tryptic
digests from parental EIAV and P 2 - 1 ....................110
vi
ABSTRACT
The polypeptide composition of purified radioactively labeled
equine infectious anemia virus (EIAV) was investigated using
high-resolution sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and guanidine hydrochloride gel filtration
(GHC1-GF) to catalog key biochemical properties including molecular
weights, stoichiometry, isoelectric points and modifications
(glycosylation or phosphorylation). The results of these studies
indicate that EIAV contains four major nonglycosylated proteins (p26,
ppl5, pll, and p9) and two glycoproteins (gp90 and gp45), which
together account for greater than 95% of the total virion protein.
Four minor polypeptides (p70, p61, p30, and p23) of unknown
significance were also detected reproducibly.
Comparisons of the
major purified proteins by peptide mapping procedures demonstrated the
unrelatedness of the proteins, including two glycoproteins, gp90 and
gp45.
Localization experiments indicated that gp90 and gp45 constitute
the envelope glycoproteins while nonglycosylated polypeptides (p26,
ppl5, pll and p9) constitute the internal components of the virus.
EIAV pll is closely associated with the viral genome in a
ribonucleoprotein complex and ppl5 seems to be a component of inner
coat while p26 and p9 appear to comprise the icosahedral core shell.
Thus, our results demonstrated that EIAV, a putative lentivirus,
contains polypeptides analogous to the structural polypeptides of
prototype-C oncornaviruses (Montelaro and Bolognesi, 1980).
vii
The recurrent nature of equine infectious anemia (EIA) has been
attributed to relatively rapid antigenic variations in EIAV during
persistent infection under selective immune pressures.
This model was
tested by biochemical analyses of five serologically distinct EIAV
isolates, recovered from experimentally inoculated ponies (Orrego,
1983).
Comparative SDS-PAGE analysis of proteins and two-dimensional
peptide mapping of their tryptic digests revealed that gp90 and gp45
undergo structural alterations during virus replication.
No
differences were found in the internal antigens after virus replicated
in a single animal; however, EIAV p26 did indicate a few structural
variations when the virus was passaged into another animal.
Thus, serological differences among EIAV isolates are correlated
to the structural variations in the polypeptides of EIAV.
The
capacity to alter antigenic structure, especially envelope
glycoproteins, may represent an important mechanism for retroviral
persistence in the presence of immune response by the host.
viii
CHAPTER I
INTRODUCTION
EQUINE INFECTIOUS ANEMIA, THE DISEASE
Equine infectious anemia (EIA) is an acute hemolytic disease of
horses, caused by a nononcogenic retrovirus (Archer et al., 1977;
Charman et al., 1976; Cheevers et al., 1977; Nakajima et al., 1969,
1970; Tajima et al., 1969).
Historically one of the first diseases
shown to be caused by a virus (Vallee and Carre, 1904), it has been
known for about 80 years.
Progress has been made only within the last
several years toward understanding the disease at the cellular and
molecular level.
Colloquially called swamp fever, it occurs in the
members of the family equidae (Issel and Coggins, 1979).
In the
United States, Louisiana has the highest reported incidence of the
disease.
From 1976 to 1981, about 5% of the horses sampled in this
state tested positive for EIA, but in some areas of Louisiana the
infection rate may be as high as 30 to 40% (Foil and Issel, 1982).
Clinical Symptoms
EIA is unique among retrovirus infections because of its episodic
character (Crawford et al., 1978; Issel and Coggins, 1979; Orrego et
al., 1982).
Following infection, the disease is not progressive, but
occurs during unpredictable "cycles", between which the horse may be
clinically normal.
Clinical symptoms and associated bursts of viremia
occur in sequential episodes, separated by several weeks or months.
The clinical response of horses following artificial inoculation or
natural exposure to equine infectious anemia virus (EIAV) is variable
1
2
and depends, in part, on host resistance factors, viral virulance and
environmental factors.
Horses developing the clinical disease EIA
have been described as acute, subacute or chronic cases.
Acute Cases - Acute EIA is most often associated with the first
exposure to the virus and results from massive virus replication in
infected macrophages and their eventual destruction.
include fever and hemorrhages.
Clinical signs
Neither anemia nor edema are seen at
this stage.
Subacute and Chronic Cases - Horses with the chronic form of EIA
have unpredictable periodic episodes of illness during which more
classical clinical signs of EIA, such as loss of weight, anemia,
subcutaneous edema, lethargy, leukopenia and depression of central
nervous system are observed (Orrego et al., 1982).
Many of the clinical signs in horses with chronic EIA may be due
to changes induced by immune reactions to viral products (Henson and
McGuire, 1971).
The anemia observed during disease may be explained
on this basis, although pathogenesis of the anemia has not been
elucidated.
Since EIAV possesses a hemagglutinin (Sentsui and Kono,
1976; Sentsui and Kono, 1981), virus particles may attach to
erythrocytes, and specific immunoglobulins then attach to viral
proteins.
The immune complexes on the cell surface would then attract
the complement.
The complement coated erythrocytes are found in
anemic EIAV-infected horses (Perryman et al., 1971), and the affected
erythrocytes have a short life span and are phagocytized by leukocytes
(McGuire et al., 1969).
Occasionally, affected horses undergo the initial febrile attack
and remain asymptomatic for the remainder of their lives.
3
Nevertheless, they harbor the virus for life.
A high percentage of
infected horses exhibit no clinical symptoms associated with
infection, although the virus or the viral products could be detected
by agar gel immunodiffusion (AGID) test (see below) in blood of such
animals (Kemen and Coggins, 1972; Issel et al., 1982).
Such
apparently healthy but infected animals seem to play an important role
in the spread of this virus by interrupting blood feeding insect
vectors (Foil and Issel, 1982).
Transmission
Transmission of EIA is mainly horizontal and rarely vertical.
Horizontal transmission may result from hematophagous arthropod
insects, feeding on horse blood (Foil et al., 1982; Issel and Coggins,
1979; Williams et al., 1981), or from use of contaminated instruments
or syringes while transferring infective blood or blood products
(Issel and Coggins, 1979).
The potential for transmission from an
infected donor appears to be higher in the presence of clinical signs
than in absence.
This seems to be directly correlated with the level
of free virus in the donor's blood and tissues.
The virus can be
demonstrated in the secretions and excretions of horses with acute
EIA.
The virus apparently can cross the placental barrier and cause
fetal infection.
However, more than 75 percent of the foals from
infected mares can be raised free of EIA (Foil and Issel, 1982; Kemen
and Coggins, 1972).
Diagnosis
EIA can be diagnosed on the basis of its typical clinical signs,
hematology, pathology or by specific laboratory tests.
Clinical signs
4
are not always specific, hence more specific laboratory tests are
important and useful in the diagnosis of this disease.
Laboratory
tests involve the detection of virus or EIA specific antibody in the
suspected animal.
One of the tests demonstrating the presence of EIAV in the blood
of a horse is the horse inoculation tests and is carried out by
injecting its blood into a susceptible horse.
Development of disease
in the recipient animal conclusively demonstrates the presence of the
virus.
However, the procedure is expensive and time consuming and,
under the best circumstances, it is often difficult to interpret the
clinical signs.
A number of serological tests have been used in research and in
the diagnosis of EIA infection.
These include complement fixation
test (Kono and Kabayashi, 1966), serum neutralization test and agar
gel immunodiffusion (AGID) test (Coggins et al., 1972; Roth et al.,
1971).
of EIA.
The AGID test has been the most useful test in the diagnosis
It detects the presence of EIA specific antibody in the serum
of the animal.
It is reliable and reasonably sensitive.
A
correlation between precipitating antibody and EIA viremia has been
demonstrated by Coggins et al. (1972).
However, as with any
serological test, the AGID test does not detect the phase of infection
prior to antibody production.
More sensitive serological tests, such as radioimmunoassay
(Coggins et al., 1978), immunofluorescence (Crawford et al., 1971) and
enzyme linked immunoassay (Montelaro et al., 1982), have been utilized
to detect EIAV antigen or antibody.
They can aid in diagnosis in the
cases in which AGID titers are extremely low.
5
Prevention or treatment of EIA has not yet been successful
because of our lack of understanding of the disease and the causative
agent, EIAV, at the molecular level.
RETROVIRUSES
Although retroviruses have been isolated from a diverse group of
animals and can produce a vast array of pathological consequences
through a variety of mechanisms resulting in oncogenic as well as
nononcogenic diseases, they have several characteristics in common.
These include the nature of their genomes, their means of entering
cells, their composition and structure, and their mode of replication
via a DNA-intermediate. The last property distinguishes this class of
viruses from other RNA viruses.
As a result, viruses that use
virus-coded, RNA-directed DNA polymerases to replicate their RNA
genomes are grouped together as "retroviruses". The common taxonomic
features of Retroviridae are summarized in Table 1-1.
Morphological Features
The structure of retroviruses has been investigated by electron
microscopy (Gross, 1970; Vigier, 1970).
Most retroviruses are roughly
spherical, about 100 nm in diameter (Bernhard et al., 1958).
They
comprise a core enclosed in an outer envelope made of a unit membrane,
with spikes and knobs projecting from the outer surfaces (Figure 1-1).
These spikes/knobs are made of glycoproteins (Rifkin and Compans,
1971).
By virtue of their morphology, as revealed in the electron
micrographs, retroviruses can be classified into four categories:
A-type, B-type, C-type and D-type particles (Bernhard, 1960; Fine and
Schochetman, 1978; Sarkar et al., 1972).
Table 1-1:
Taxonomic features of Retroviridae
Morphology
i
Spherical enveloped virions (80-120 nm diameter),
variable surface projections (8 nm), icosahedral
capsid containing a ribonucleoprotein complex
Physico­
Density 1.16 to 1.18 g/ml in sucrose, 1.16 to
chemical
1.21 g/ml in cesium chloride, sensitive to lipid
properties
solvents, detergents, and heat inactivation (56°C,
30 min); highly resistant to UV and X-radiation
Nucleic acid
Linear positive-sense single-stranded RNA (60 S-70 S)
composed of 2 identical subunits (30 S-35 S); 5' cap
(M^G^ppp^NpNp); polyadenylated 3' end; 1% by weight
Protein*
About 60% by weight; gag, internal structural proteins
(4-5); pol, reverse transcriptase (1-2); env, envelope
proteins (1-2)
Lipid
About 35% by weight; derived from cell membrane
Carbohydrates
About 4% by weight, associated with env proteins
+ Teich (1982)
* The figures in the brackets indicate numbers of polypeptides.
7
A-type particles occur as intracellular forms only and do not
have any infectivity.
They are 60-90 nm in diameter, with an
electron-luscent center surrounded by a double shell.
B-type particles have two distinguishing morphological features.
First, long spikes are seen on the cell surface at budding site with
doughnut-shaped cores, 75 nm in diameter, underneath.
Second, after
budding, the mature particles contain electron-dense nucleoids that
are eccentrically located within the enveloped particle.
The
prototype member of the B-type particles is mouse mammary tumor virus
(MMTV) (Table 1-1).
A key feature of C-type viruses is the absence of
intracytoplasmic viral structures until budding has commenced at the
plasma membrane.
Crescent-shaped electron dense cores are seen at the
plasma membrane at specific sites.
also be seen.
Spikes of the virion envelope can
As virus maturation proceeds, the core eventually
develops into a sphere with an electron-luscent center, and the cell
membrane begins to pinch off as it surrounds this structure.
Finally,
extracellular forms with centrally located electron-luscent (immature)
or electron-dense (mature) cores are observed.
The majority of
retroviruses isolated to date are of the C-type morphology including
oncornaviruses.
D-type particles are associated with both intracellular and
extracellular forms.
Intracellular particles are ring-shaped,
60-95 nm in diameter, and are generally most abundant near the plasma
membrane (Immature D-type particles). Mature D-particles are
100-120 nm in diameter and have an electron-dense core that is
8
eccentrically located (Manning and Hackett, 1972).
The virus
classified in this class is Mason-Pfizer monkey virus (MPMV).
Subfamilies
The family Retroviridae has been classified further into three
subfamilies, namely, oncovirinae, lentivirinae and spumavirinae. The
main criteria for this classification have been cross-reacting
group-specific major antigen within the subfamily and the
characteristic disease the virus causes in an infected animal.
Oncovirinae includes the oncogenic and related nononcogenic
retroviruses, the isolates that are difficult to classify in a
retrovirus subfamily, and the noninfectious retrovirus-like particles.
Lentiviruses are viruses which cause slow infections,
characterized by a prolonged incubation period and a protracted
symptomatic disease phase.
The diseases caused by lentiviruses in
sheep are chronic pneumonia, maedi (shortness of breath) and visna
(wasting due to progressive neurological impairment, with inflammation
of central nervous system). The subfamily consists of one isolate
from goats (caprine encephalitis arthritis virus) and several isolates
from sheep, including visna, maedi, and progressive pneumonia virus
(PPV). Because of the chronic nature of EIAV-induced disease, EIAV is
often considered a lentivirus, however the absence of antigenic
relatedness to visna virus (Stowring et al., 1979) does not
substantiate this classification.
Spumaviruses or foamy viruses have been isolated from a number of
mammalian species, including man, and establish persistent infections
without evidence of pathogenesis.
These viruses induce a
characteristic spontaneous foamy degeneration of cells after a few
9
weeks in culture.
They share the common features of retroviruses.
Typical spumaviruses are simian foamy virus (SFV) (Rabin et al., 1976)
and bovine syncytial virus (BSV) (Malmquist et al., 1969).
Properties of type-C Onconaviruses
Biochemical and serological properties of type-C oncornavirus
structural components have been reviewed recently (Montelaro and
Bolognesi, 1980; Schafer and Bolognesi, 1977).
Common general
features of the structural proteins of type-C viruses are discussed
here for the purpose of subsequent comparison with polypeptides of
EIAV and other lentiviruses.
The spikes and knobs observed on the lipid envelope of the virion
are composed of one or two glycoproteins and one or none
nonglycosylated proteins coded by the env gene of the virus genome
(Figure 1-1).
Underneath the lipid envelope is the inner coat structure
containing a single polypeptide, which is usually phosphorylated. The
inner coat surrounds the icosahedral core, which is made up of at
least two major structural proteins.
Inside the icosahedral core
is a
viral RNA genome in helical form with associated reverse transcriptase
molecules and a very basic structural polypeptide.
These inner
structural polypeptides are coded by the gag gene and are group
specific or type specific antigens.
Thus, analyses of oncornavirus proteins have consistently led to
the identification of four or five small nonglycosylated polypeptides
with molecular weight ranging from 10,000 to 30,000 daltons, and one
or two larger glycoproteins.
10
Figure I—1:
A model of retroviruses indicating the structural
components.
KN = knob, VM = viral membrane, IC = inner coat,
CS = core shell and RNP = ribonucleoprotein complex (Montelaro
and Bolognesi, 1980).
11
Prototype-C Oncornaviruses
Friend murine leukemia virus (FLV) and Rous sarcoma virus (RSV)
are prototype-C oncornaviruses representing mammalian and avian type-C
viruses, respectively.
Morphologically these viruses are quite
similar and have analogous structural components (Montelaro and
Bolognesi, 1980).
The polypeptide compositions of FLV and RSV were
determined using high resolution sodium dodecyl sulfate (SDS)
polyacrylamide gel electrophoresis (PAGE) and guanidine
hydrochloride-gel filtration (GHC1-GF) (Montelaro et al., 1978).
Qualitative and quantitative properties of the virion structural
components and their position in the intact virion, deduced from
localization experiments described below, are summarized in Table 1-2.
Localization of Structural Polypeptides
Virion polypeptides can be assigned to certain site(s) in the
intact virus particle and in relation to each other by a variety of
direct and indirect techniques.
These include differential labeling,
protease treatment, preparation of viral substructures and use of
cross linking reagents.
The surface proteins comprising the spikes and the knobs of the
virion can be specifically radioiodinated using lactoperoxidase
(Cheevers et al., 1980; David and Reisfield, 1974; Phillips and
Morrison, 1971) or can be removed on careful digestion of the whole
particles with protease (Mosser et al., 1977; Montelaro et al., 1982).
They constitute the surface knobs and spikes, visible in the electron
micrographs and are responsible for many biological properties of the
viruses (Schafer and Bolognesi, 1977), including host cell specificity
and induction of neutralizing antibody.
The knob structure is
12
Table 1-2:
Structural components of avian and murine type-C
retroviruses
Virion polypeptide
Avian
Murine
Structural component
Virion structure
gp85(ll%)
gp71(10%)
Knob
gp35(9%)
pl5E(2%)
Spike
pl0(5%)
pl2E(13%)
Envelope associated
pP19(ll%)
ppl2(7%)
inner coat
inner coat
p27(36%)
p30(50%)
core shell
core
pl5(13%)
pl5C(13%)
core associated
exterior
pl2(13%)
pl0(4%)
nucleoprotein
Ribonucleo-
p91(8)(1%)
p70(a)(1%)
reverse transcriptase
protein
p64(a)(1%)
Montelaro and Bolognesi (1980).
►
Envelope
complex
13
believed to be composed of the hydrophilic envelope glycoprotein (gp85
or gp71), while the
spike contains the hydrophobic envelopeproteins,
like avian gp35 and murine pl5E,
bilayer.
which are believed to span the lipid
These latter proteins aggregate in 6 M guanidine
hydrochloride and require detergents to keep them in solution
(Montelaro et al., 1978).
These surface knob and spike components may
or may not be linked by intermolecular disulfide linkages depending on
the individual virus studied.
The retroviral particles can be partially disrupted, using
controlled concentrations of nonionic detergents (Coffin and Temin,
1971; Davis and Rueckert, 1972; Teramoto et al., 1977) to generate
subviral particles.
Polypeptide analysis of subviral particles on
SDS-PAGE and electron microscopic observation can allow an assignment
of the polypeptide to the different structural features of the virion.
Mild detergent treatment of
retroviruses releases core structures
that band in equilibrium sucrose gradients at a density of
approximately 1.24-1.26 g/ml, compared with intact virions that band
at 1.16-1.18 g/ml (Teramoto et al., 1977).
The increase in density is
due to the solubilization of the lipid containing envelope and to
release of the internal core structure.
Electron microscopic studies
on virus and core structures show that the murine C-type cores possess
icosahedral symmetry (Nermut et al., 1972; Schafer and Bolognesi,
1977).
SDS-PAGE analysis of the cores show that they contain the most
abundant nonglycosylated polypeptide found in virions (usually with a
molecular weight of 24,000-30,000 daltons) and a smaller, highly basic
polypeptide.
14
Further detergent treatment of the cores releases the genomic RNA
associated with the basic polypeptide (Bolognesi, 1973; Davis and
Rueckert, 1972) and reverse transcriptase.
In avian viruses, the
basic component is designated as pl2 and, in murine viruses, plO
(Bolognesi, 1974).
Electron microscopic investigations with murine and avian type-C
viruses (Frank et al., 1978) revealed the presence of a thin layer,
designated the inner coat, located just beneath the viral envelope as
demonstrated in the virus model (Figure 1-1).
Analogous major
phosphoproteins, ppl9 in avian and ppl2 in murine viruses, are
believed to comprise the inner coat structure.
These phosphoproteins
are neither present in purified virus core nor detectable on the virus
surface (Schafer and Bolognesi, 1977).
Also in avian viruses ppl9
could be cross-linked to gp35 when intact virions were treated with
reversible cross-linking reagnets.
The phosphoproteins are the most
type specific antigens of the respective viruses (Bolognesi et al.,
1975; Hunsmann et al., 1976; Schwarz et al., 1976) and exist in
phosphorylated as well as nonphosphorylated forms (Erickson et al.,
1977; Lai, 1976; Pal and Roy-Burman, 1975; Sen et al., 1976, 1977).
They also have the affinity for the homologous viral RNA (Leis et al.,
1978; Sen et al., 1976, 1977; Sen and Todaro, 1977).
Replication of Retroviruses
Retroviruses are unified by the manner in which their genomes are
replicated during infection.
In its simplest form, replication can be
envisioned as
RNA
DNA -> RNA
with the first step mediated by a virus-coded, RNA-directed DNA
15
polymerase (reverse transcriptase) carried in the infectious
particles, and the latter step mediated principally by host enzymes,
also responsible for expression of cellular genes.
Retroviruses usually associate stably with the cells they infect.
This is accomplished by covalent integration of retroviral DNA copy
into the chromosomal DNA of host cells.
Intermediate proviral DNA
molecules synthesized from viral RNA are partial double stranded
linear DNA, closed circular molecules and finally, the integrated
provirus (Coffin, 1979).
A simplified schematic view of the major
events in the replication of a retrovirus are presented in Figure 1-2
(Varmus and Swanstrom, 1982).
Transcription of proviral DNA results
in the synthesis of at least two types of mRNA, a larger one serves as
a message for gag and pol gene products while the smaller mRNA codes
for env proteins.
The large RNA species is also the viral genome
which is packaged into the virus.
EQUINE INFECTIOUS ANEMIA VIRUS
As mentioned before, EIA is one of the first diseases shown to
have a viral ("filtrable") etiology.
The wide range of pathogenic
properties of this disase can be attributed to single retrovirus,
called the equine infectious anemia virus (EIAV). Apparently, the
infected animal mounts an immune response against the invading virus,
usually 16-42 days post-infection.
virus completely.
However, it does not eliminate the
Once infected, the virus persists in the host.
Some animals experience periodic bursts of viremia at unpredictable
intervals (Kono et al., 1973; Orrego et al., 1982), and they are
usually associated with characteristic symptoms of EIA described
earlier.
Such a periodic disease process is unique among retrovirus
16
Figure 1-2:
genome.
A simplified representation of replication of retrovirus
1.
Viral RNA;
= 5' and 3 1 terminal repeats;
= 5' and 3' untranslated regions; PB = primer binding site; G,P,
and E = gag, pol, and env genes, respectively.
and double stranded DNA synthesis.
viral DNA.
5.
4.
2 and 3.
cDNA
Circular double stranded
Integration of viral DNA into host genome.
Transcription of proviral DNA to yield mRNAs and viral RNAs.
6.
17
Rs U5PB
*5
G
P
E
I
I
I
US P b
U 3 R3,
u, r# u,pB
e
u, rs us
1
03,
a
s
mnm
u , r 5u 5 p b
m
4
" o'
I
g
p
e
u , r , u t.
//////////<
G
I
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E
18
infections and raises a number of intriguing questions concerning the
molecular biology of EIAV.
The most important problems posed by EIAV are
clinical disease and bursts of viremia periodic and
a) why is the
b) where and how
does the virus persist in the presence of an apparently active immune
response?
Both of these questions must be viewed in the light of our
knowledge about other retroviruses, but they also require an
understanding of the unique biochemical and immunological properties
of EIAV.
Growth Characteristics
Whereas the clinical and epidemiological aspects of EIA have been
exhaustively dealt with and amply reviewed (Crawford et al., 1978;
McGuire and Henson, 1973), knowledge of the molecular biology of EIAV
had been slow due to the lack of a suitable tissue culture system for
obtaining the virus in the quantities required for characterization.
EIAV multiplication was apparently restricted in cells’cultured in
vitro, with the exception of equine leukocyte cultures (Kabayashi and
Kono, 1967).
However, the lack of a convenient method of measuring
the virus propagation in these cultures and the presence of high
concentration of serum required for growth and maintenance of
leukocyte cultures complicated the purification of EIAV.
EIAV strains
have recently been selected for replication in equine fibroblast
cultures without cytopathic effects (Kono and Yoshino, 1974; Malmquist
et al., 1973).
These laboratory strains now permit cultivation,
propagation and purification of EIAV in quantities sufficient for
biochemical and immunological analysis.
That these laboratory strains
are representative isolates of EIAV is indicated by the facts that
19
they retain their virulence for horses after greater than 50 passages
in equine fibroblasts, and retain their ability to replicate in equine
fibroblasts in vitro after 3 clinical cycles in vivo (Crawford et al.,
1978).
The isolate of EIAV described by Malmquist et al. (1973) grows
in monolayer cultures of an equine dermal cell line and primary equine
embryonic spleen.
Subsequently, they precipitated the virus from
culture fluids with polyethylene glycol, removed the viral lipid with
ether, and assayed the antigen using a radial immuno-diffusion
technique.
It has since been noted (Matheka et al., 1976) that the
virus, adapted by Malmquist and coworkers to grow in equine dermal
cells and fetal spleen, propagates equally well in other equine tissue
culture systems, including primary equine fetal lung and kidney cell
cultures.
In vitro and in vivo Replication of EIAV
The cell adapted strain of Malmquist et al. (1973) has been
routinely grown in tissue culture systems by a number of workers for
biochemical characterization (Cheevers et al., 1976; Matheka et al.,
1977; Sentsui and Kono, 1981) and morphological studies (Matheka et
al., 1976; Weiland et al., 1977).
Anderson et al. (1979)
characterized the in vitro infection of equine dermal fibroblasts and
demonstrated that the cells persistently infected with EIAV show no
alterations in cell morphology or growth kinetics when compared to
uninfected cells.
The production of extracellular virus was shown to
be uniform throughout the cell cycle.
Also the normal levels of
progeny virus were produced in cultures treated with mitomycin C (DNA
synthesis inhibitor) and placed in serum-containing medium.
This was
in contrast to oncornaviruses which require that host cells pass
20
through at least one cell cycle to initiate viral RNA synthesis and
subsequent virus production (Bader, 1975; Fischinger et al., 1975;
Paskind et al., 1975).
These results suggest that either the
integration of EIA proviral DNA is independent of host DNA synthesis
or the integration of provirus, which does occur in equine dermal
(Crawford et al., 1978) and kidney (Rice et al., 1978) fibroblasts, is
not a prerequisite to replication.
An independence of viral
replication and host cell DNA synthesis, if true, is one of the
characteristics that EIAV shares with visna virus, a lentivirus
(Thormar, 1976), a relationship that may carry important pathogenic
implications.
In contrast to the restrictive host range iri vitro, after
inoculation of EIAV in a pony, there is rapid virus proliferation to
high titers in practically all tissues (Kono et al., 1971).
Most of
the infectivity in the plasma is in the form of infectious
virus-antibody complexes (Henson and McGuire, 1974).
Between febrile
episodes, infectivity declines in both serum and tissues, the liver
and spleen retaining higher titers than other organs.
Proviral DNA of EIAV
The persistent nonlytic infection of equine fibroblasts with EIAV
is maintained by the integration of proviral DNA into the cellular
genome (Crawford et al., 1978; Rice et al., 1978) which could be
detected by reassociation study with cDNA synthesized from the viral
RNA.
This revealed an average of two to six viral DNA copies per
haploid genome (Cheevers et al., 1982; Rice et al., 1978).
There was
no detectable hybridization of EIAV cDNA with normal horse DNA,
showing that it is not an endogenous virus of the horse.
Sequences
21
related to EIAV were neither found in DNA of other Equus species, nor
in a variety of other mammals.
Also EIAV cDNA did not hybridize with
a variety of other retroviruses, thus showing no sequence homology.
Morphological and Physicochemical Characteristics
EIAV has been classified as a member of the family Retroviridae
(Charman et al., 1976) because of morphological features and
biochemical properties it shares with other retroviruses.
Studies on
EIAV indicate that the particles have a buoyant density of
1.15-1.16 g/ml at 20°C.
(Charman et al., 1976; Matheka et al., 1976;
Sentsui and Kono, 1981) and the sedimentation constant, S2Q w » for the
virus averages 656 ± 5S.
Morphologically, particles are 80-120 nm in
diameter with a very flexible but usually spherical shape (Matheka et
al., 1976; Teich, 1982; Weiland et al., 1977).
Inside the particles
are pleomorphic, spherical or rod-shaped electron dense nucleoid,
40-60 nm in diameter or length.
Thin surface projections or knobs
have been observed on the outside, and sometimes an inner shell around
the core is seen (inner coat). EIAV buds from the plasma membrane
following the appearance of a crescent-shaped nucleoid under the
plasma membrane, typical of C-type particles.
Thus, EIAV displays a
complex morphology characteristic of type-C viruses (Gonda et al.,
1978).
EIAV also shares other characteristics with the retroviruses.
These include sensitivity to ether (Nakajima and Obara, 1964),
sensitivity to IdU during the early stages of replication (Kono et
al., 1970), 60-70 S RNA genome (Charman et al., 1976; Cheevers et al.,
1977) made of two identical single stranded 34S subunits and a reverse
22
transcriptase enzyme associated with the virions (Charman et al.,
1976; Cheevers et al., 1978).
EIAV is a non-oncogenic virus and has epidemiological
similarities to the viruses of slow infections, lentiviruses.
However, the genomic and serological unrelatedness to either
oncoviruses or lentiviruses has not permitted further definitive
classification of EIAV into a retrovirus subfamily.
Structural Components of EIAV
A prerequisite to the detailed study of any virus and associated
disease is the knowledge of the structural components of the virus.
EIAV is a retrovirus with protein, lipid and nucleic acid content
typical of other retroviruses.
Proteins comprise about 60% of the
virion mass and are important components for structural and functional
reasons.
Immune responses of the animal are directed against these
proteins.
A thorough description of the major antigens of EIAV is
fundamental to understanding the dynamic interaction between host
immune mechanism and the virus antigens.
However, prior to this
study, little definitive work was available on the structural
polypeptides of EIAV.
Since serological comparisons have failed to detect any
relatedness between EIAV and a number of other retroviruses (Charman
et al., 1976; Stowering et al., 1979), this has led to the suggestion
that the polypeptide composition of EIAV may be distinct from type-C
retroviruses (Charman et al., 1976; Rice et al., 1978).
Preliminary
descriptions of EIAV polypeptide composition have been contradictory
and thus have been unable to resolve the exact relationship of EIAV to
other retroviruses.
23
The major nonglycosylated protein of EIAV, which is employed in
standard immunodiagnostic tests, is apparently a group specific
antigen with a molecular weight of 25,000-29,000 (Barta and Issel,
1978; Cheevers et al., 1977; Ishizaki et al., 1976) although one
report claims a molecular weight of only 7,600 (Hart et al., 1976).
Cheevers et al. (1978) reported further that 80% EIAV structural
protein is accounted for by five polypeptides, including two
nonglycosylated components (p29 and pl3) which comprise one-half of
the virion protein and three glycoproteins (gp77/79, gp64 and gp40).
They also describe eight or nine minor polypeptides of unknown
significance.
Ishizaki et al., (1978) detected three major
nonglycosylated proteins (p25, pl4 and pll), but only two
glycoproteins (gp80 and gp40). While both of the above investigators
employed SDS-PAGE as the sole analytical procedure, Charman et al.
(1979) used isoelectric focussing to detect four major nonglycosylated
components [p25, pl2, plO (acidic) and plO (basic)], but made no
statements concerning virion glycoproteins.
Thus the exact
polypeptide composition of EIAV, and the relationship of the
polypeptides to each other and to analogous polypeptides of other
retroviruses remains to be determined.
ANTIGENIC VARIATION
An invading organism, as a result of multiplication in the host,
induces the host to elicit an immune response.
A strong immune
response, normally, will eliminate the invading organism and thus cure
the host.
However, successful parasites have devised ways to evade
the host immunosurveillance system and survive.
24
The host elicits a neutralizing antibody directed against
antigens responsible for infectivity.
located on the surface.
Usually these antigens are
In the case of viruses these surface antigens
or proteins are important for cell recognition, attachment and
infection.
A parasite that can vary its surface antigens, at a
sufficient rate, will present itself in a form new to the host and
will not be eliminated.
This type of phenomenon involving alterations
in structural antigens of the parasite during replication in an animal
is called antigenic variation.
Antigenic variations in surface
components only are likely to benefit the parasite in its survival
against the host immune response.
Multiplication or replication of
the parasite with altered surface antigen will then occur, until an
effective host immune response against this strain ensues.
Several
parasites, including some protozoan and a few virus members of some
families, undergo antigenic variation.
Trypanosomes
Trypanosomes are parasitic protozoa which cause sleeping sickness
in man and animals, primarily in developing countries (Goodman, 1978;
Walsh and Warren, 1979).
These tsetse fly-transmitted parasites
inhabit the blood stream and tissue spaces of their mammalian hosts,
where they rapidly stimulate the production of trypanocidal antibody.
Before all the trypanosomes can be eliminated, however, antigenically
distinct variants appear in the bloodstream which perpetuate the
infection, until they, in turn, are removed by the antibody.
Usually,
the trypanosomes can elude the immune system in this cycle, which
leads eventually to the death of the host.
25
The antigenic profile of the trypanosome is governed by the
structure of its surface coat.
This coat is made up of a densely
packed layer of glycoprotein molecules, the variant surface
glycoproteins (VSGs), whose structure is unique for each variant
antigen type.
The genome contains more than 100 variant antigen genes
probably generated by gene duplication and mutation (Englund et al.,
1982), and by controlling the expression of these genes, the
trypanosome is able to parade a sequence of different variants past
the immune system of the host.
Poliovirus
Antigenic variation has been observed in poliovirus, a
picornavirus.
The attenuation of neurovirulent poliovirus involves
multiple variations in the single stranded RNA genome and in its gene
products (Kew et al., 1980), resulting in nonvirulent oral
poliovaccine strains.
The currently used poliovaccine strains were
selected as spontaneous mutants of wild isolates following multiple
passages in simian extraneural cells and tissues (Sabin and Bougler,
1973).
The surfaces of wild type and attenuated viruses could be
distinguished by serological tests (Van Wezel and Hazendonk, 1979).
Structural alterations were detected in three of the four viral
structural proteins, VP1, VP3, and VP4, and it was estimated that 1.5%
of total amino acids were substituted in the course of attenuation
leading to the vaccine strain.
Genetic changes observed here are essentially of the same nature
that can also lead to a reversion of an attenuated, nonvirulent strain
to a virulent one.
Indeed, multiple genetic changes (>100) have been
found to occur in the attenuated virus after the ingestion of oral
26
poliovaccines and subsequent replication of virus in humans (Kew et
al., 1981; Nottay et al., 1981).
These virus isolates were recovered
subsequent to the vaccine related cases of poliomyelitis.
SDS-PAGE,
peptide mapping and oligonucleotide mapping were used to detect
changes in proteins and genomes, respectively.
It was demonstrated
that both the gene and gene products of poliovirus evolve at
detectable rates during replication in humans.
This is in sharp
contrast to what happens during propogation in cell culture, where the
RNA genome and the viral proteins are remarkably conserved.
mediators for change in a virus population are antibodies.
Obvious
However,
other factors must play a role, because changes in the virus
polypeptides were not restricted to the virion structural proteins,
where alterations might be expected to occur in response to antibody
pressure, but were also found in the non-capsid proteins, which are
thought to function only intracellularly (Kew et al., 1981).
Probably, the dynamics of selection are very complex because the virus
population is highly mutable and the human population is
immunologically heterogeneous.
In any case, the attenuated poliovirus undergoes genetic
variation upon human intestinal passage and there is a potential for
these viruses to revert to virulence as a result of multiple genetic
variation.
Influenza Virus
In contrast to picornaviruses like poliovirus, which contain
positive-strand single stranded RNA, influenza viruses contain a
segmented negative-strand RNA genome.
Influenza viruses are unique in
their potential to cause epidemics and pandemics and are characterized
27
by a high degree of genetic variability.
These viruses are divided
into three types, A, B and C, based on differences in their internal
proteins.
They also differ in their biological and epidemiological
behaviour (Young et al., 1980). Most influenza cases in man are caused
by type A viruses which are further divided into subtypes according to
serological differences of their surface antigens, hemagglutinin (HA)
and neuraminidase (NA). Although there are antigenic subtypes among
other DNA and RNA viruses, these strain variants are rather stable in
nature and there is no evidence of such constant modification of viral
genome and viral proteins as it occurs among influenza viruses.
Two
types of antigenic variations are observed among these viruses,
antigenic drift and antigenic shift.
Antigenic drift is a gradual change observed in the surface HA
and NA proteins among the virus isolates and is caused by mutational
changes in the viral genome leading to single amino acid changes (Both
et al., 1980; Laver et al., 1980) and selective pressure from immunity
in human populations.
The drift is usually slow.
In laboratory,
variants were recovered from an infected animal and they were found to
contain changes in peptide maps of the HA subunit (Laver and Webster,
1972).
Antigenic shift causes the advent of new pandemic viruses that
are very different from the strains prevalent before or during the
period the new virus emerged.
These changes take place invariably in
HA and sometimes also in NA antigen.
shift is not clearly understood.
(Nayak, 1977).
The mechanism of the antigenic
Two hypotheses have been proposed
28
The direct mutation theory maintains that antigenic shift is an
extension of antigenic drift in which accumulated mutation and
selection pressure eventually produce a new strain different enough to
cause a pandemic.
However, the differences between strains
responsible for subsequent pandemics are so numerous that it is
difficult to visualize how the mutations could have taken place all at
once.
The genetic recombination theory proposes that gross changes are
caused by genetic exchange among influenza A viruses prevalent in
nature.
Since influenza viruses contain a segmented RNA genome,
recombination may take place with high frequency following
co-infection of a cell with two viruses.
Such recombinants or hybrid
viruses are produced by mixed infection in cell culture, in eggs as
well as in animals (Webster, 1972).
Also many of the viruses from
different species share common antigens, indicating occurrence of
recombination.
Visna Virus
Visna virus, a retrovirus, is the etiologic agent of an
inflammatory and degenerative disease of the central nervous system of
the sheep (Brahic and Haase, 1981; Hasse, 1975).
The disease is
characterized by a prolonged incubation period, followed by a subacute
progression of symptoms to death.
The unusual aspects of visna is the
life-long persistence of the virus which undergoes progressive
antigenic variation in a single infected animal (Narayan et al., 1977;
Narayan et al., 1978).
Biochemical characterizations of such variants
recovered from a sheep revealed alterations in the genome when
examined by oligonucleotide mapping to separate RNase T1 resistant
29
fragments (Clements et al., 1S80).
Alterations in the genome were
localized near the 3' end, presumably in the gene coding for envelope
products.
The structural alterations in the envelope glycoprotein,
gpl35, could be detected by two dimensional peptide mapping,
whilethe
internal proteins were found to be conserved (Scott et al.,
1979). By
altering the envelope glycoprotein, visna virus is believed to evade
the host immunosurveillance system.
However, recently Thormar et al.
(1983) reported the presence of originally inoculated virus in the
experimentally infected animal, long after inoculation, and thus
questioned the importance of antigenic variation in visna.
The Case for Antigenic Variation in EIAV
The periodic nature of equine infectious anemia (EIA) has been
observed by several investigators (Kono et al., 1971; Kono et al.,
1973) and recently these observations were confirmed by Orrego et al.
(1982).
Horses experimentally inoculated with a "cloned" virus
exhibited a chronic disease with periodic relapses of fever and
associated viremia.
Kono and coworkers (1973) recovered virus
isolates during successive febrile episodes and carried out serum
neutralization assays demonstrating that virus isolates were not
susceptible to previously formed neutralizing antibodies but reacted
with antibodies produced some weeks later.
Also the late isolates
differed from one another when examined in cross-neutralization tests.
Immune sera used did not have high specificity for a particular virus,
but this may not be surprising considering the presence of some common
antigenic determinant and the heterogeneous immune response of the
host.
The results did strongly indicate that occurrence of virus
variants in vivo as the most likely explanation for their resistance
30
to heterologous antisera.
Moreover, these isolates were found to be
genetically stable upon tissue culture passages.
The variants could be differentiated from one another by
neutralizing antibodies, but not by complement fixing and
precipitating antibodies (Kono et al., 1971).
These results suggest
that the differences among the variants are in their surface antigens.
Since the host elicits neutralizing antibody against these surface
antigens, the structural alterations in these molecules can have
significance with regards to antigenic variation in EIAV.
A model for the chain of events following infection of a horse
with EIAV is presented in Figure 1-3.
This antigenic variation
appears to play a crucial role in the establishment of a persistent
infection and also presents a major obstacle in developing a
vaccination protocol for this economically important disease.
RESEARCH OBJECTIVES
The epidemiology of the EIA is very interesting as demonstrated
by its unpredictable periodic occurrence and associated bursts of
viremia.
An apparent breakdown of the control mechanisms which
suppress virus replication between the cycles allows a resurgent
viremia.
This outpouring of virus and associated antigens in the face
of a vigorous immune response sets in motion a series of
immunopathologic events that cause the clinical signs and the lesions
of EIAV.
Several questions remain unanswered concerning the mechanism
of the disease and the molecular biology of EIAV.
The present
investigation has attempted to answer some of the questions regarding
the molecular biology of EIAV, and these are listed below along with
their significance.
31
C H R O N I C EIA
virus 1
immune response
to virus 1
virus 2
immune response
to virus 2
T I ME
Figure 1-3:
A schematic view of the sequence of events in an animal
suffering with chronic EIA.
32
a.
What is the polypeptide composition of EIAV?
Is it unique and
distinct from the polypeptide composition of other retroviruses which
apparently do not undergo antigenic variation during replication in a
single animal?
These studies were designed to provide a definitive
analysis of EIAV protein composition and to form a basis for further
study of EIAV antigens.
b.
What are the properties of structural polypeptides of EIAV and how
are these polypeptides related to each other and to the polypeptides
of other retroviruses?
The answers to these questions would establish
the relationship of polypeptides, and help to understand the functions
of these components.
c.
How are these polypeptides organized in the intact virion?
The
knowledge of the polypeptide organization in EIAV would enable us to
compare EIAV with the current retrovirus model and help focus our
attention on polypeptides which could be important for antigenic
variation.
d.
What is the biochemical nature of antigenic variation?
This
question is fundamental to understanding the existence of variants in
a single animal and the role of antigenic variation in virus
persistence.
No vaccine protection is available yet against this economically
important disease because we do not know enough about the EIAV
antigens and the mechanism of antigenic variation.
It is hoped that
the investigation carried out here will bring research closer to
development of a vaccine against EIAV and will also further our
knowledge of antigenic variations in viruses.
CHAPTER II
MATERIALS AND METHODS
Reagents
A list of chemicals used throughout this study, including
abbreviations and sources of supply, is shown below.
acetic acid - Mallinckrodt, Inc.; acetone - MCB; acrylamide Biorad; ammonium bicarbonate - Sigma; ammonium persulfate - MCB;
ampholite (pH 3-10) - LKB; autofluor - National Diagnostics; blue
dextran - Sigma; bovine serum albumin, crystalline (BSA) - Sigma;
bromelain, pineapple, grade II - Sigma; bromophenol blue - Sigma;
n-butanol - Baker; cellulose coated thin layer plates, 20 x 20 cm - EM
Lab; chloramine-T - Sigma; p-chloromercuriphenylsulfonate - Sigma;
chymotrypsin - Sigma; coomasie brilliant blue - Sigma; 7-deoxycholate
(sodium salt) (DOC) - Sigma; 2 ,4-dinitrophenyl alanine (DNP-alanine) Sigma; diphenyl carbamyl chloride treated-trypsin (DPCC-trypsin) Sigma; Eagle's minimal essential medium (MEM) - GIBCO Lab.;
ethylenediamine - Sigma; ethylene dinitrilo tetraacetic acid (EDTA) MCB; fetal calf serum - GIBCO Lab.; formic acid -MCB/Baker; guanidine
3
- Sigma; [ H]-glucosamine - NEN; glycine - Sigma; glycerol - J.T.
Baker; hydrochloric acid - MCB; hydrogen peroxide - Sigma; isopropanol
- MCB; lactoperoxidase - Calbiochem; lentil lectin Sepharose 4B -
1A
3
C]-leucine - NEN; [ H]-leucine - NEN; 2-mercaptoethanol
Pharmacia; [
- Eastman; 3-mercaptopropionate - Sigma; methanol - Mallinckrodt;
N,N'-methylene bisacrylamide - Biorad; new born calf serum - GIBCO
Lab.; nonidet P-40 (NP-40) - Particle Data Laboratories, Ltd.;
penicillin G - Sigma; phosphoric acid - MCB;
32
P-phosphoric acid -
34
ICN; pyridine - Mallinckrodt, Inc.; Sepharose 6B - Pharmacia; sodium
azide - MCB; sodium bicarbonate - Sigma; sodium chloride Mallinckrodt, Inc.; sodium dodecyl sulfate (SDS) - Biorad; sodium
hydroxide - J.T. Baker; sodium iodide - Mallinckrodt, Inc.;
125
125
I-sodium iodide (
I) - NEN; sodium metabisulfite - MCB; sodium
phosphate, dibasic - Sigma; sodium phosphate, monobasic - Sigma;
streptomycin sulfate - Sigma; sucrose - MCB; N,N,N',N'-tetramethylene
diamine (TEMED) - Biorad; trichloroacetic acid (TCA) - MCB;
tris(hydroxymethyl) aminomethane (Tris) - Sigma; Urea - Sigma; V8
protease (Staphyloccoccus aureus) - Miles Lab.
Virus Propagation and Purification
The cell-adapted Wyoming strain (Malmquist et al., 1973) of EIAV
was propagated in primary equine fetal kidney cells maintained in
Eagle's minimal essential medium (MEM) supplemented with 4 to 10%
sterile newborn or fetal calf serum, 25 mM HEPES buffer, 10 mM sodium
bicarbonate, 150 U of penicillin G per ml, and 150 yg of streptomycin
sulfate per ml (Amborski et al., 1979).
Persistently infected
cultures were maintained in roller bottle cultures containing 50 ml of
medium.
Under these conditions virus production continued for
approximately 3 months, until cell senescence.
Supernatant medium was harvested every 3 to 4 days for virus
purification.
After the cell debris was removed from the medium by
centrifugation at 8000 rpm for 30 min in a Sorvall GSA rotor
(8500 x g), the virus containing supernatant was concentrated about
10- to 40-fold by using a Pellicon membrane filter cassette system
(nominal exclusion 10^ daltons molecular weight) (Millipore Corp.,
Bedford, Mass.).
The virus concentrate was then layered over a 10-ml
35
cushion of 10% sucrose, and the virus was pelleted by centrifugation
in a type 19 rotor at 19,000 rpm (50,000 x g) for 2 h.
The pelleted
virus was suspended in 10 mM sodium phosphate buffer, pH 7.2 and
sedimented to its equilibrium density (1.18 g/ml) on 30 ml 20 to 80%
(v/v) glycerol gradients in a Beckman SW28 rotor centrifuged at
28,000 rpm (130,000 x g) for 3 h.
4°C.
All procedures were carried out at
After the run, the virus band was withdrawn with a syringe.
With these procedures, about 10 mg of EIAV (estimated as protein,
Lowry et al., 1951) could be purified per week from 10 roller bottles
yielding one liter of tissue culture fluid (Montelaro et al., 1982).
Virus Isolates
For biochemical comparisons, EIAV isolates were obtained by
serial passage of the Wyoming strain of EIAV in Shetland ponies
(Orrego et al., 1982).
and isolate number.
Isolates recovered were designated by passage
For example, isolate Pl-1 was recovered from
plasma of a first-passaged pony (No. 47), two weeks after the
inoculation when the pony became AGID test positive.
Isolates P2-1
and P2-6 were obtained from plasma samples of a second-passaged pony
(No. 82) during the first and sixth febrile episodes, respectively.
Isolates P3-1 and P3-3 were obtained from plasma samples of a
third-passaged pony (No. 127) during first and third febrile episodes,
respectively (Figure II-l and Figure 111-13).
For the biochemical studies presented here, the viruses recovered
from end-point dilutions of respective plasma samples were propagated
in fetal equine kidney cells and purified by standard procedures.
Parental
first
passage;
EIAV
PI -1
second
passage:
*
P2-1
third
passage:
*
P3-i-^ -*-P3-3
Figure II-l:
P2-6
Scheme indicating the generation of EIAV isolates in
experimentally inoculated ponies.
37
Radioactive Labeling of EIAV in Vivo
a.
[3H]-leucine or [^C]-leucine-EIAV
Infected fetal equine kidney cells in falcon flasks were
transferred overnight to leucine-free medium.
Radioactively labeled
EIAV was propagated in leucine-free medium containing 25 pCi/ml of
[3H]-leucine or 2.5 pCi/ml of [^C]-leucine.
3
b. [ H]-glucosamine-EIAV
Infected fetal equine kidney cells were transferred overnight to
medium reduced to l/10th the normal glucose concentration.
3
[ H]-glucosamine-EIAV was propagated in the same medium supplemented
3
with 25 pCi/ml of [ H]-glucosamine.
c.
[32P]-EIAV
32
The virus was labeled with [ P] as described by Pal et al.
(1975).
Briefly, the labeling was carried out by growing the infected
fetal equine kidney cell monolayers in 50% phosphate-free MEM
supplemented with 10% dialyzed fetal calf serum and 100 pCi/ml of
[32P].
After 24 h intervals of growth in the appropriate radioactive
medium, the virus containing media were centrifuged to remove the
cellular debris, and the virus was purified from the supernatant fluid
by centrifugation procedures essentially as described before
(Montelaro et al., 1978).
Radioactive Labeling of the Virus or the Virus Components in Vitro
a.
125
I-surface labeling of EIAV using lactoperoxidase
Procedures for surface specific radioiodination using
lactoperoxidase have been described (Phillips and Morrison, 1971;
David and Reisfield, 1974).
About 40-75 pg of purified unlabeled EIAV
38
was suspended in 0.5 ml of PBS-A, pH 7.2 and the following were added
in the given order:
125
1 mCi of
1(20 yl), and 1 unit of
lactoperoxidase (50 units/ml in PBS-A).
addition of 10 yl of ^©£(10
The reaction was initiated by
—2
—2
M). Three subsequent additions of 10
^2^2 were ma(*e at 5 min intervals.
M
At the end of a 20 min period, the
reaction was terminated by adding 1.4 ml of PBI containing 0.02%
sodium azide.
The diluted reaction mixture was then layered onto a
glycerol step gradient (20%:80%::2ml:lml) and centrifuged at
30,000 rpm (100,000 x g) for 60 min at 4°C in a SW50.1 rotor.
The
gradient was then fractionated into BSA-coated plastic beakers, and
5 yl from each fraction was counted for radioactivity in a Packard
gamma counter.
Fractions containing
125
I-labeled EIAV were pooled,
diluted with 0.01 M phosphate buffer and pelleted by centrifugation in
SW50.1 rotor at 45,000 rpm (240,000 xg) for 45 min.
The pellet was
suspended in cold 0.01 M phosphate buffer, pH 7.2 and stored at -80°C.
b.
125
I-labeling of virus polypeptides using chloramine-T (Hunter,
1978).
The following solutions were prepared fresh in 0.05 M sodium
phosphate buffer, pH 7.4: chloramine-T, 0.4%; sodium metabisulfite,
0.25%; and sodium iodide, 1%.
To a 10 x 75 mm test tube, 25-50 yg of
protein solution was added, followed by 0.05 M phosphate buffer,
pH 7.4 to a volume of 200 yl and 1 mCi of
125
I (20 yl). The
iodination was initiated by addition of 25 yl of chloramine-T.
The
reaction was allowed to proceed for 60 sec and then terminated by
adding 100 yl of sodium metabisulfite.
binding of
125
To minimize the noncovalent
I to proteins, 200 yl of carrier sodium iodide solution
was added and mixed well.
39
Unreacted
125
I was separated from radiolabeled protein using a
column (0.8 x 22 cm) of Sephadex G-25 prepared in a disposable 10 ml
pipet and eluted with PBS-A.
c.
125
I-labeling of polypeptides in gel slices using chloramine-T.
For certain purposes (e.g. peptide mapping), it was convenient to
radioiodinate proteins contained in gel slices after SDS-PAGE
separation (see below).
The method has been described by Elder et al.
(1977b), and adapted as follows.
Protein bands were detected by
staining with 0.25% Commassie brilliant blue for 30 min, followed by
destaining in 7.5% acetic acid (v/v) and 25% methanol (v/v).
The
protein bands of interest were then excised from gel with a clean
scalpel.
Gel slices, contained in perforated wells of a microliter
plate, were washed with 25% isopropanol for 4 to 6 h and then with 10%
methanol overnight to remove SDS and other contaminants.
The gel
slices were then placed in siliconized tubes and dried under a heat
lamp and vacuum.
The proteins were radioiodinated with
125
I in the
dried gel slices by a modification of the chloramine-T method
(Greenwood et al., 1963).
The following components were added to each
tube containing the dried gel slice:
1 mCi of
125
I (20 pi),
50yl of
0.5 M sodium phosphate buffer, pH 7.5 and, finally, 10 yl of
chloramine-T solution (0.5%) in phosphate buffer.
After a 30 min
reaction time, the labeling was stopped by the addition of 1 ml of
sodium metabisulfite (0.1%).
This was followed by extensive washing
of the gel slices with 10% methanol to remove noncovalent
as possible.
125
The gel slices were once again dried as before
preparation for enzyme digestion (see below).
I asmuch
in
Proteins contained in
40
the gel slices could be labeled by this procedure to a specific
activity of about 5 x 10^ cpm/pg of protein.
High Resolution Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis and Analysis of Gels
The procedure used for sample preparation, sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE) on slab or
cylindrical gels and analyses of gels by staining, fluorography or
fractionation have been described (Montelaro and Bolognesi, 1978;
Shealy and Reuckert, 1978; Shealy et al., 1980).
Gels used were 10% polyacrylamide and contained 0.1% SDS, 0.1%
N,N,N',N'-tetramethylenediamine, 0.5 M or 1.0 M urea and 0.1 M sodium
phosphate, pH 7.2.
Slab gel dimensions were 14 cm x 28 cm x 1.5 mm.
The cylindrical gels were 0.6 cm in diameter by 20 to 25 cm in length.
The electrophoresis buffer was 0.1 M sodium phosphate, pH 7.2 and
contained 0.1% SDS and 10 mM 3-mercaptopropionate.
Aqueous samples in 10 mM phosphate buffer pH 7.2, containing
unlabeled or radioactive proteins, were made 1% in SDS and
2-mercaptoethanol each in a total volume of 100-200 pi and were heated
in a boiling water bath for 3 min.
Sucrose was added to a final
concentration of 20 to 30%, and 1 pi of 0.5% bromophenol blue in
0.02 N sodium hydroxide was added to each sample.
Samples were
layered onto the prerun gels and were electrophoresed at 50 mA per
slab gel and at 8 mA per tube gel for about 18-20 h.
Changes in pH
were prevented by circulating the buffer between upper and lower
buffer chambers during electrophoresis.
41
For non-reducing SDS-PAGE analysis, 2-mercaptoethanol was omitted
from the sample preparation, and 3-mercaptopropionate was excluded
from running buffer.
When samples contained non-radioactive proteins, gels were
stained with 0.25% Coomassie Brilliant Blue dye in acetic acid:
methanol: water (92:454:454, v:v:vD) for 1 h and destained extensively
with 7.5% (v/v) acetic acid and 25% (v/v) methanol in water to reveal
the protein bands.
For analysis of radioactive samples on tube gels, an opaque
"crushing plug" of highly cross-linked polyacrylamide (40%) was
polymerized on top of the experimental gel after electrophoresis, and
the gels were crushed bottom first into scintillation vials with a
Gilson automatic fractionator (Gilson et al., 1972).
After overnight
elution of radioactive fractions into water, 2 ml of tritosol was
added to each fraction, mixed with and counted in a scintillation
counter.
Radioactive samples analyzed on slab gels were revealed by
autoradiography or fluorography of the dried gel depending on the
isotope used.
3
14
Fluorography was performed for [ H] or [ C] after
treatment of the slab gel with autofluor (National Diagnostics) for
1-2 h.
(Shealy et al., 1980).
Discontinuous SDS-PAGE
For some experiments, e.g. peptide analysis after limited
proteolysis by the method of Cleveland et al., (1977), the
discontinuous gel system described by Laemmli (1970) was used.
A 15%
acrylamide slab gel (acrylamide: biscrylamide:: 40:1) containing 10%
SDS, 0.08% TEMED and 1% ammonium persulfate was prepared in 0.375 M
42
Tris-HCl buffer, pH 8.8.
A 4% stacking gel (3 to 4 cm in height) was
overlayered on the 15% separating gel.
Samples (50-100 pi) were prepared in a disruption buffer
consisting of 0.0625 M Tris-HCl buffer, 0.2% SDS, 5%
2-mercaptoethanol, 6 M urea and 10% glycerol, adjusted to pH 6.8 and
heated 100°C for 3 min.
The electrophoresis buffer was 10 mM
Tris-glycine with 0.1 SDS, pH 8.4.
Runs were carried out at 35 mA for
15-18 h.
Guanidine Hydrochloride Gel Filtration
The procedures used for sample preparation and the conditions
used for guanidine hydrochloride (GHC1) gel filtration (GF) were based
on the methods of Green and Bolognesi (1974) and Montelaro et al.
(1978, 1982).
Routine procedures for solubilizing retrovirus protein
samples in guanidine hydrochloride incompletely dissociate the
proteins of EIAV, some of which then chromatograph together in the
void volume as aggregates.
However, it was found that EIAV proteins
can be resolved more efficiently if the virion lipids are extracted
with acetone before the proteins are treated with guanidine
hydrochloride (Montelaro et al., 1982).
Thus, in a typical
preparative experiment about 30 mg of purified EIAV in 4 ml of 10 mM
sodium phosphate buffer, pH 7.2 was mixed with 10 volumes of cold
acetone and incubated at 0-4°C for 45 min with frequent mixing.
The
resulting protein precipitate was collected by centrifugation in a
Sorvall SS34 rotor at 10,000 rpm (10,000 x g) for 30 min, and the
supernatant acetone phase containing viral lipid was removed.
protein pellet was then solubilized in 4 ml of 8 M guanidine
hydrochloride in 50 mM Tris buffer, pH 8.5, containing 2%
The
43
2-mercaptoethanol by heating in a boiling waterbath for 5 min and at
45°C for 45 min.
Any insoluble material was removed by another
centrifugation, but usually quantitative solubilization of the
starting virus protein was achieved.
Dextran blue and DNP-alanine
were added to the final concentrations of 0.08% and 0.025%,
respectively.
The solubilized proteins were then analyzed on a column
(1.5 x 95 cm) of Sepharose 6B eluted with 6 M guanidine hydrochloride
in 20 mM sodium phosphate buffer, pH 6.5, at a flow rate of 1.2 to
1.6 ml/h.
The recommended sodium acetate buffer, pH 5.0 (Green and
Bolognesi, 1974), was not used as an eluting buffer, because it
resulted in the rapid degradation of Sepharose matrix.
The absorbance
of the eluant was monitored at 280 nm by a continuous flow monitor,
while fractions of about 0.5 ml were collected.
The appropriate
fractions were pooled, dialyzed for 48 h against several changes of
100 mM ammonium bicarbonate, and finally dialyzed against 50 mM
bicarbonate for 24 h.
The dialyzed samples were lyophilized to
dryness and suspended in an appropriate buffer.
A recovery of 60 to
70% of the initial virus protein was usually observed at this stage of
the purification.
The radioactive virion proteins were isolated in similar manner
from radiolabeled virus.
6
3
Typically, 3 x 10 cpm of [ H]-leucine or
14
[ C]-leucine-EIAV was used for a preparative run.
Aliquots from the
collected fractions were counted for radioactivity, after adding 2 ml
of tritosol.
Radiolabeled protein fractions were pooled, dialysed and
lyophilized as described above.
44
Polypeptide Nomenclature and Quantitation
Two procedures, GHC1-GF and SDS-PAGE, have been particularly
effective in elucidating retrovirus polypeptide composition and have
therefore been chosen as a basis for deriving a nomenclature for
virion polypeptides (August et al., 1974).
Thus, a glycoprotein was
designated by gp, and nonglycosylated polypeptide by p and a
phosphorylated polypeptide by pp.
This was followed by a number which
indicated the apparent molecular weight of polypeptide in thousand
daltons as estimated by one of the two techniques mentioned above.
For polypeptides of molecular weight below 30,000 daltons, estimation
was based on the results of GHC1-GF.
For polypeptides of molecular
weight above 30,000 daltons, estimation by SDS-PAGE was chosen for
nomenclature purposes.
Individual virion polypeptides were quantitated from SDS-PAGE
3
analysis of [ H]-leucine-EIAV. Percentages of total radioactivity
were calculated for each of the major and minor virion polypeptides by
summing the radioactivity in each protein peak.
three GHC1-GF runs were used to average data.
Three SDS-PAGE and
Total radioactivity was
the sum total of all the counts observed in the polypeptide peaks.
From the knowledge of apparent molecular weight for each of these
polypeptides, the numbers of polypeptide chains per virion were
g
calculated assuming a virion protein mass of 4 x 10
daltons as
described by Davis and Rueckert (1972).
Calculations were made in two ways.
First, the assumption was
made that leucine is present in virion polypeptides in equivalent
amounts.
A second set of calculations resulted from the knowledge of
45
the number of leucine residues present in each of the major internal
polypeptides of EIAV (Montelaro et al., 1982).
Polypeptide Characterization
a.
Isoelectric focussing
Protein isoelectric points were determined by using cylindrical
polyacrylamide gels (0.6 x 10 cm) containing 7.5% acrylamide, 0.2%
bisacrylamide, 5 M urea, and 1.6% ampholine (pH range 3.5 to 10, LKB
Munich, Germany).
Protein samples were dissolved in deionized water
containing 5 M urea and 10% sucrose.
Electrolyte solutions consisted
of 1% ethylenediamine at the anode and 1.4% phosphoric acid at the
cathode.
Electrophoresis was carried out at 4°C at constant power (3
watts) until less than 1 mA of current was observed (usually 10-12 h).
Gels were then fractionated into 1 mm fractions with an automatic gel
fractionator (Gilson Medical Electronics, Middleton, WI) (Gilson et
al., 1972), and the fractions were assayed for pH and radioactivity
(Montelaro et al., 1978, 1982).
b.
Phosphoprotein analysis
Purified
32
P-EIAV was analyzed by standard high resolution
SDS-PAGE and GHC1-GF procedures.
such analyses of
32
Since viral RNA incorporates
32
P,
P-EIAV were made before and after treatment with
RNase to identify the phosporylated polypeptide(s) without ambiguity.
For RNase treatment
32
P-EIAV was disrupted in 20 mM Tris-HCl
buffer, pH 7.4, containing 0.1% Nonidet P-40 at 0°C for 1 h and then
treated with RNase (0.5 mg/ml final concentrations) at 37°C for 2 h.
c.
One-dimensional peptide mapping
The method used to generate peptides by limited proteolysis in
SDS and their analysis by SDS-PAGE was essentially that of Cleveland
et al., (1977).
3
Purified, homogeneous preparations of [ H]-leucine
labeled internal proteins of EIAV (p26, ppl5, pll, and p9) were taken
in sample buffer which contained 0.125 M Tris-HCl at pH 6.8, 0.5% SDS,
10% glycerol and 0.0001% bromophenol blue.
100°C for 2 min.
The samples were heated to
Proteolytic digestions were carried out at 37°C for
30 min by addition of given protease (chymotrypsin or
protease) at the final concentration of 1 Ug/10 yl.
aureus V8
Proteolysis was
stopped by boiling the samples for 2 min after addition of
2-mercaptoethanol and SDS to final concentrations of 10% and 2%,
respectively.
The resultant peptides were then analyzed by electrophoresis in
gels containing SDS in a slab gel apparatus (Studier, 1973) utilizing
the discontinuous system described earlier (Laemmli, 1970). After the
3
run, [ H]-leucine labeled peptides were detected by fluorography of
the dried gel.
Proteases used in this experiment were quite specific in their
action.
Chymotrypsin specifically cleaves peptide bonds involving a
carbonyl group from an aromatic amino acid (e.g. Tyr, Trp and Phe).
On the other hand, V8 protease from Staphylococcus aureus cleaves
bonds with carbonyl group from glutamic acids, and sometimes from
aspartic acid.
d.
Two-dimensional peptide mapping
Procedures for two-dimensional peptide mapping have been
described by Elder et al., (1977a,b).
Radioiodinations of individual
polypeptides in gel slices, after separation by SDS-PAGE, were carried
out as described earlier.
heat lamp and vacuum.
Subsequently, gel slices were dried under a
DPCC-treated trypsin (30 ug = 210 units) was
47
then soaked into the gel, and 0.5 ml of 0.05 M ammonimum bicarbonate
(pH 8.0) was added.
Digestion was allowed to proceed at 37°C for
20-24 h, when most of the digested peptides eluted out of the gel.
The solution containing the tryptic peptides was lyophilized to
dryness and the residue was dissolved in 20 to 50 yl of
electrophoresis solvent (acetic acid: formic acid: water:: 15:5:80).
Peptides were separated on cellulose coated thin layer plates, first,
by electrophoresis at 1000 volts.
The movement of the peptides was
monitored by 1 yl of dye mixture (27, Orange G and 1% acid fuschin)
spotted at the diagonal corner.
After the run, the plate was
completely dried, and separation in perpendicular direction was
accomplished by chromatography using freshly prepared n-butanol:
pyridine: acetic acid: water (6.5:5:1:4).
125
I-labeled peptides were
then revealed by standard autoradiographic techniques.
Polypeptide Localization Experiments
a.
Bromelain digestion
Virion surface and internal proteins were differentiated based on
their susceptibilities to protease digestion when intact
3
[ H]-leucine-EIAV was treated with bromelain. Bromelain (pineapple)
was prepared for use by making a suspension of 150 mg of bromelain
powder in 1.5 ml of 20 mM sodium phosphate buffer, pH 7.2, with 0.15 M
NaCl containing 0.5 mM p-chloromecuriphenysulfonic acid.
This
reversible inhibitor was included to prevent autodigestion of
bromelain during storage.
The suspension was clarified by
centrifugation for 20 min at 4000 rpm (1500 x g) in a Sorvall SS34
rotor.
The large, pale yellow pellet was discarded.
The clear
supernatant containing crude bromelain was diluted to 4 mg/ml with the
48
same buffer, stored at -80°C, and used as the stock solution for
subsequent experiments.
Bromelain digestion of EIAV was carried out by a modification of
the procedure of Rifkin and Compans (1971), which was used for the
structural analysis of spleen necrosis virus (SNV) by Mosser et al.
(1975).
In a typical experiment, 100 pi of [^H]-leucine-EIAV
(300,000 cpm) was treated with 100 pi (=15 units) of bromelain stock
solution and 200 pi of 0.2 M Tris-HCl buffer (pH 7.2) containing 1 mM
EDTA and 20 mM 2-mercaptoethanol.
The digestion mixture was incubated
for 2.5 to 3 h and was then diluted to 1 ml with 10 mM Tris-HCl,
pH 8.1, containing 1 mM EDTA, 0.3 M NaCl and 0.1% BSA and subsequently
chilled.
The chilled mixture was layered onto a 3.2 ml 10 to 20%
(w/v) sucrose gradient underlayered with a 1.0 ml 50% (w/v) sucrose
cushion.
Sucrose solutions were in the same buffer that was used to
dilute the digestion mixture.
The gradients were centrifuged for 1 h
at 30,000 rpm (100,000 x g) and 4°C in a Beckman SW50.1 rotor.
Fractions were collected through a needle puncturing the bottom of the
tube, and the virus peak was located by determining the radioactivity
of samples of each fraction.
Peak fractions were pooled, diluted with
10 mM Tris-HCl, pH 8.1, 1 mM EDTA, 0.3 M NaCl and 0.1% BSA, and the
virus was pelleted by centrifugation at 45,000 rpm (240,000 x g) for
45 min in SW50.1 rotor.
b.
Pelleted virus was then analysed by SDS-PAGE.
Virion surface radioiodination using lactoperoxidase.
Virion surface and internal proteins were also differentiated
based on their susceptibilities to surface radioiodination using
lactoperoxidase.
The method for radioiodination has been described in
an earlier section.
49
c.
Production of a subviral particle (ribonucleoprotein).
A subviral particle (ribonucleoprotein, RNP) of an apparent
density 1.31 g/cm3 was prepared by treatment of virus with Nonidet
P-40 (NP-40, a nonionic detergent) and dithiothreitol (DTT) as
reported by Davis and Rueckert (1972) and Mosser et al. (1975) with
some modifications.
A 4 ml continuous gradient was prepared from 30%
sucrose in 10 mM Tris-1 mM EDTA, pH 8.1, with 0.4% dithiothreitol and
65% sucrose in the same buffer, made with D2O.
About 5 mg of EIAV was
treated with 1 ml of 0.5% NP-40 and 1% DTT in 10 mM Tris-1 mM EDTA
buffer, pH 8.1, at 4°C and layered immediately onto the above chilled
sucrose gradient and centrifuged at 45,000 rpm (240,000 x g) and 4°C
for 3 h in Beckman SW50.1 rotor.
Fractions were collected by
puncturing the tube at the bottom with a needle.
In a parallel
3
analytical run made with [ H]-leucine-EIAV, fractions were assayed for
radioactivity and protein peaks corresponding to disrupted viral
proteins and subviral particle were detected.
This helped to locate
the appropriate fractions from the gradient containing the
nonradioactive EIAV.
Protein peaks were pooled and treated with cold
10% TCA to precipitate the proteins.
The precipitated proteins were
pelleted by centrifugation at 10,000 rpm (10,000 x g) for 20 min in
SS34 rotor.
Pellets were washed with chilled acetone and finally
dried under nitrogen.
Pellets were suspended in 10 mM phosphate
buffer, pH 7.2, to be analyzed by SDS-PAGE as described earlier.
Glycoprotein Purification and Analysis
a.
Lentil lectin affinity chromatography.
Procedures for lectin affinity chromatography of EIAV have been
described and evaluated previously (Monetlaro, et al., 1983).
50
Typically, about 2 mg of EIAV was treated with ten volumes of cold
acetone to remove viral lipids and to preciptate viral proteins, which
were collected by centrifugation at 10,000 rpm (10,000 x g) for 10 min
at 4°C in Sorvall SS34 rotor.
The dried protein pellet was
solubilized in buffer A (20 mM Tris-HCl, 100 mM NaCl, pH 8.3)
containing 0.5% 7-deoxycholate (sodium salt) (DOC) and incubated at
37°C for 30 min with intermittant vortexing and brief episodes of
sonication.
It was then centrifuged at 10,000 rpm for 10 min in SS 34
rotor to remove insoluble material.
Solubilized proteins were loaded
on a Lens culinaris (lentil) lectin Sepharose column (1-2 ml bed
volume), preequilibrated with buffer A containing 0.1% DOC.
Nonbinding proteins were eluted when washed with the same buffer.
The
column was then washed with buffer A-0.1% DOC containing 0.2 M
a-methylglucoside to remove bound glycoproteins.
Nonbinding and
binding proteins were detected by monitoring the column effluent at
280 nm.
Appropriate fractions were pooled, dialyzed against 100 mM
NH^HCOg and lyophilized.
The protein residues were then dissolved in
10 mM phosphate buffer, pH 7.2 and were separated by SDS-PAGE on 7.5%
acrylamide slab gels as described earlier,
b.
Glycoprotein Variation Study
Isolated glycoproteins from prototype virus and other EIAV
isolates were suitable for radioiodination and for further analysis by
two dimensional peptide mapping procedures of Elder et al., (1977a,b),
which enabled us to detect the minor differences between the two or
more proteins.
Details of these procedures have been described in
earlier sections.
51
In order to assay for variation in the glycosylated regions of
the virion glycoproteins the following strategies were used.
First,
3
[ H]-glucosamine labeled glycoprotein (gp90) was isolated and purified
3
by a preparative SDS-PAGE of [ H]-glucosamine-EIAV.
The purified
3
[ H]-glucosamine-gp90 was then incubated with DPCC-trypsin, and the
resultant peptides were separated on cellulose coated thin layer
plates by two-dimensional peptide mapping procedure, as described
before.
Secondly,
125
I-labeled peptides of gp90, prepared as described
earlier, were separated into glycosylated (binding) and
nonglycosylated (non-binding) components by lentil lectin affinity
chromatography.
The separated peptide pools were dialyzed against
100 mM NH^HCO^ using Spectrapor 3 dialysis tubing, which was boiled in
10 mM EDTA, pH 8.0 to reduce the exclusion limit to 700 daltons (Dr.
Blackeney, personal communication).
The peptides were then
lyophilized to dryness and separated by conventional two-dimensional
peptide mapping.
CHAPTER III
RESULTS
I.
ANALYSES OF POLYPEPTIDES OF EIAV
14
The polypeptides of EIAV were analyzed using [ C]-leucine or
[^H]-leucine labeled EIAV, [^H]-glucosamine labeled EIAV and
unlabeled EIAV, prepared and purified as described in Chapter II.
Previous experience with retroviruses has consistently emphasized the
need to utilize several analytical techniques in identifying virion
structural polypeptides (Montelaro et al., 1978).
In this study, EIAV
polypeptides were analyzed by the complementary techniques of high
resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE), and guanidine hydrochloride gel filtration (GHC1-GF) on a
column of Sepharose 6B (Parekh et al., 1980; Montelaro et al., 1982),
as described earlier.
SDS-PAGE ANALYSIS
Radioactive EIAV was analyzed on tube gels which were
fractionated after the run to determine the position of radioactivity.
A representative electrophoretic radioactivity profile of purified
14
[ C]-leucine labeled EIAV is presented in Figure III-1A.
3
t H]-leucine labeled Friend murine leukemia virus (FLV), a prototype-C
oncornavirus, was analyzed simultaneously for comparative purposes and
as a molecular weight calibration standard.
FLV proteins are denoted
in italics (gp71, p30, pl5E, pl5C, pl2, pl2E and plO), while EIAV
proteins are labeled in block letters (gp90, p70, p61, gp45,
52
Figure III-l:
SDS-PAGE analysis of radioactively labeled EIAV (A)
[^C]-leucine EIAV (----) vs. [^H]-leucine FLV (--- ) (B)
3
[ H]-glucosamine EIAV.
54
S D S - P A G E ANALYSIS
P 'S E
P 12
p15C
nI
*
a
o.
u
z
0
3
w
J
1
u
DISTANCE MIGRATED (mm)
■O
1 2
a
<L
u
(A
o
0
3
9P * 5
e
1
x
n
50
100
DISTANCE
ISO
MIGRATED (mm)
200
55
p30, p26, p23, ppl5, pll and p9), designated according to the
nomenclature system proposed by August et al. (1974) (see below).
SDS-PAGE analysis of EIAV reveals four major low-molecular weight
polypeptides, p26, ppl5, pll and p9, and also resolves reproducibly
several high-molecular weight components.
These include two
polypeptides of 90,000 (gp90) and 45,000 (gp45) daltons molecular
weight, which are glycosylated (see below) and four minor
non-glycosylated proteins designated as p70, p61, p30 and p23.
3
[ H]-leucine labeled FLV, simultaneously resolved on the same tube
gel, gave an analogous radioactivity profile (broken line) with four
major low molecular weight proteins, p30, pl5, pl2 and plO; and a few
high-molecular weight components, including a glycosylated protein,
gp71 (Montelaro and Bolognesi, 1978).
To identify which of the above EIAV polypeptides were
3
glycosylated, [ H]-glucosamine labeled EIAV was propagated in fetal
equine kidney cells, purified and analyzed by SDS-PAGE in tube gels
(Figure III-1B) as described above for radioactive leucine labeled
3
EIAV. The radioactivity profile of [ H]-glucosamine-EIAV shows two
distinct peaks which coincided with polypeptides of apparent molecular
weight 90,000 and 45,000 daltons.
These proteins, being glycosylated,
were designated as gp90 and gp45.
Frequently, EIAV pll migrates as a shoulder to ppl5, and they do
not separate on SDS-PAGE.
A similar problem has been encountered
during the polypeptide analysis of spleen neicosis virus (Mosser et
al., 1975), especially in gels of leucine labeled virus.
Use of 1 M
urea, instead of 0.5 M, resulted in enhanced separation of EIAV ppl5
and pll.
56
GHC1-GF Analysis
Purified radioactive virus was analyzed by GHC1-GF as described
for FLV by Montelaro et al. (1978).
The chromatographic radioactivity
14
profile of [ C]-leucine labeled EIAV (solid line) is presented in
3
Figure III-2A with a simultaneous analysis of [ H]-leucine FLV used
for comparison and as a molecular weight calibration standard (broken
line).
FLV proteins are indicated in italics (gp71, p30, pl5, pl2 and
plO) and EIAV proteins are labeled in block letters (gp90, p26, ppl5,
pll and p9). The void volume is represented by a radioactive peak,
designated Vo, while DNP-alanine serves as the dye marker for the
total column volume.
The chromatographic analysis of the radioactive EIAV reveals six
distinct peaks of radioactivity.
As observed with other
oncornaviruses, the first protein(s) to elute from GHC1-GF are
contained in the void volume fractions of the column indicating an
apparent molecular weight of at least 100,000 daltons.
The next EIAV
protein(s) elutes as a relatively broad peak with an apparent
moleuclar weight of about 74,000.
This component is followed by the
major EIAV structural polypeptide (p26) which displays a molecular
weight of 26,000 and, in succession, by three additional components of
15,000 (ppl5); 11,000 (pll) and 9000 (p9) molecular weights,
respectively.
A comparison of the respective polypeptide profiles of FLV and
EIAV, shown in Figure III-2A, indicates that the two viruses contain
remarkably similar structural components.
are noted reproducibly,
However, two differences
i) the percentage of radioactivity eluting in
the void volume is higher in the case of EIAV than in the case of FLV
Figure III-2:
GHC1-GF analysis of radioactively labeled EIAV (A)
[^C]-leucine EIAV (----) vs [^H]-leucine FLV (----) (B)
3
[ H]-glucosamine EIAV.
58
GUANIDINE HYDROCHLORIDE-GEL FILTRATION (GHCI-GF)
9
p26
p1S p12 pto
8
6 S
m
"C-LEUCINE
CPM X 10
7
•o
9 X
6
I
0
PP15
5
4 au
4
u
3
* HI
3 J
1
z
z
3
to
P9
DNP-Ala
2
H*GLUCOSAMINE CPM XIO
9
4
3
•P 90
2
io
30
100
FRACTION
150
(0.5ml)
200
59
and
ii) EIAV and FLV polypeptides display somewhat different apparent
molecular weights.
3
In an analogous experiment, purified [ H]-glucosamine EIAV was
analyzed by GHC1-GF (Figure III-2B) to identify the glycoproteins in
relation to [^C]-leucine proteins of EIAV (Figure III-2A) . The
conditions of analysis were identical.
The chromatographic
3
radioactivity profile of [ H]-glucosamine-EIAV gave only two distinct
radioactivity peaks, one of which eluted at the void volume fractions,
representing apparent molecular weight in excess of 100,000 daltons.
The second peak eluted with an apparent molecular weight of 74,000.
Proteins contained in these radioactivity peaks were identified from
their subsequent SDS-PAGE analysis (see below), which indicated that
all of gp45 eluted in the void volume fractions along with part of
gp90, and the remaining gp90 was present in the second peak of
radioactivity.
SDS-PAGE Analysis of GHC1-GF Pools
It is well established that certain oncornavirus proteins (eg.
RSV pl5 and pl2) display different apparent molecular weights when
analyzed by SDS-PAGE and GHC1-GF (August et al., 1974).
Thus, to
establish a definitive correlation between the EIAV proteins resolved
by these two analytical systems, each radioactive component separated
by GHC1-GF of [^H]-leucine EIAV (cf. Figure III-2A) was isolated and
subjected to high resolution SDS-PAGE.
The results of this experiment
are presented in Figure 3 and can be compared to the electrophoretic
profile of purified EIAV shown in Figure 1A.
The data in Figure III-3
demonstrate that the GHC1-GF components designated as EIAV p26, ppl5,
pll and p9 (Figure III-2A) contain a single homogeneous peak of
60
Figure III-3:
SDS-PAGE analysis of the individual protein fractions
obtained by GHC1-GF of [ H]-leucine EIAV as described in Figure
III-2A.
A-F correspond to the six radioactive components in the
order of their elution from the gel filtration column.
61
p26
ppIS
gp90
gp45
>00 ) '
OP«0
&
p26
30.
20 .
pp15
20
10
( )
l 860
Pll
0
100
CXSTANCC MIGRATED (n n )
190
(1100)
62
radioactivity upon analysis by SDS-PAGE (Figure III-3C-F,
respectively).
In contrast, the void volume fractions from GHC1-GF
reveal a complex profile in SDS-PAGE (Figure III-3A) including the
major proteins designated gp90, gp45, p26 and ppl5 as well as certain
high molecular weight minor polypeptides.
The presence of substantial
quantities of p26 and ppl5 in the void volume fractions is surprising
and suggests that even treatment of the virus with 8 M GHC1 at 100°C
fails to completely disrupt the intermolecular interactions between
some of the EIAV polypeptides.
The presence of gp90 in the void
volume may be explained by the fact that gp90 elutes immediately after
the void volume fractions of the column (Figure III-3B), along with
several of the minor high molecular weight virion components.
On the
other hand, gp45 elutes exclusively in the void volume fractions,
apparently in an aggregated state, analogous to murine pl5E/pl2E and
avian gp35 (Montelaro et al., 1978).
The chromatographic elution pattern of the EIAV glycoproteins,
gp90 and gp45, was further confirmed when the two radioactivity peaks
3
obtained on GHC1-GF analysis of [ H]-glucosamine-EIAV (Figure III-2B)
were isolated and subjected to high resolution SDS-PAGE.
The results
of this experiment are presented in Figure 4 and can be compared to
3
the electrophoretic profile of purified [ H]-glucosamine-EIAV shown in
Figure III-1B.
Void volume fractions from the GHC1-GF analysis
(Figure III-2B) reveal some of the virion gp90 and all of the virion
gp45 upon analysis by SDS-PAGE (Figure II1-4A).
In contrast, the
second glycosylated chromatographic peak to elute from the GHC1 column
contains only gp90 as demonstrated by SDS-PAGE (Figure III-4B). Thus,
63
Figure III-4:
SDS-PAGE analysis of the individual protein fractions
3
obtained by GHC1-GF of [ H]-glucosamine EIAV as described in
Figure III-2B.
A and B correspond to the two radioactive
components resolved by gel filtration in the order of their
elution from the column.
64
2
3H-GLUCOSAMINE
cpm x 10'
gp90
gp45
5
4
3
2
50
150
100
DISTANCE MIGRATED (mm)
200
65
all of gp45 preferentially elutes in the void volume, apparently in an
aggregated form, reflecting its hydrophobic property.
Preparative GHC1-GF runs were made, in an analogous manner, to
purify the major nonglycosylated polypeptides of EIAV.
These
preparative runs demonstrated that if the EIAV proteins are
precipitated and lipids removed by ten volumes of cold acetone prior
to GHCI-GF analysis, the problem of aggregation of EIAV proteins can
be avoided with only negligible amounts of proteins eluting in the
void volume (Montelaro et al., 1982).
Also, the chromatographic analysis of unlabeled EIAV, monitoring
absorbance at 280 nm, gave a profile different form the one obtained
using leucine labeled EIAV, apparently due to differences in
absorbance by each polypeptide.
EIAV pll did not show any absorbance
at 280 nm, and this correlated with the absence of tyrosine and
tryptophan in this protein (Montelaro et al., 1982).
Relative Abundance of Polypeptides
A set of calculations for the major and minor polypeptides of
EIAV, analyzed by SDS-PAGE and GHCI-GF, is presented in Table III-l.
The first column shows the designation of each of these polypeptides,
the nomenclature being based on recommendation of August et al.
(1974).
Second column represents the average percentage of total
radioactivity found in each of these components.
This is followed by
apparent molecular weights calculated using FLV polypeptides as
molecular weight standards.
Molecular weight estimations by two
procedures differ for some of this polypeptides, but only one of the
two values is selected for nomenclature (August et al., 1974; Parekh
et al., 1980).
66
Table III-l:
g
Polypeptide
Polypeptides of radioactive leucine labeled EIAV.
Percentage
total
k
radioactivity
Apparent Molecular
weight
GHCI-GF
SDS-PAGE
Estimated
polypeptide ^
chains/virion
A
B
Major
gp90
6.0
74,000
90,000
300
-
gp45
3.0
> 100,000
45,000
300
-
p26
45.0
26,000
26,000
6,900
7,700
PPl5
28.5
15,000
15,000
7,600
5,800
pll
5.5
11,000
12,000
2,000
2,600
p9
7.5
9,000
10,000
3,300
3,200
p70
1.0
-
70,000
60
-
p61
1.0
-
61,000
70
-
p30
1.5
-
30,000
200
-
P23
1.0
-
23,000
170
-
Minor
g
Protein nomenclature based on recommendations of August et al.,
(1974).
k Calculated from the data presented in Figure III-l and III-2,
averaging percentage cpm from GHCI-GF and SDS-PAGE.
C Calculated by GHCI-GF and SDS-PAGE using FLV proteins as a molecular
weight standards.
d
8
Calculated assuming that virion protein mass is 4 x 10 daltons as
described by Davis and Rueckert (1972). Column A = Supposition is
made here that % leucine distribution in virion polypeptides is same.
Column B = Calculated with the knowledge of actual % of leucine
present in the major nonglycosylated polypeptides (Montelaro et al.,
1982).
Retroviruses are estimated to have a protein mass of 4 x 10
daltons (Davis and Rueckert, 1972).
Taking this value for the protein
3
mass of EIAV and assuming that leucine, and hence [ H]-leucine, is
evenly distributed among polypeptides, an estimate of the number of
copies of each polypeptide constituting the virion particle can be
calculated (Table III-l).
Both glycoproteins, gp90 and gp45, are
found in 300 copies each per virion, while the major nonglycosylated
proteins are present from 2000 copies to 7600 copies per virion.
Other minor polypeptides ranged from below 100 to 200 copies per
virion.
From the knowledge of the number of leucine residues present per
molecule (Montelaro et al., 1982), the corrected number of major
nonglycosylated polypeptide chains per virion can be calculated.
These are presented in the last column.
The observed differences in
stoichiometry probably reflect the inaccuracy of the assumption of an
equal distribution of leucine residues in each EIAV protein.
II.
CHARACTERIZATION OF EIAV POLYPEPTIDES
Peptide Mapping
a).
Cleveland's Limited Proteolysis (One-dimensional peptide
mapping).
Previous reports from other laboratories (Cheevers et al., 1978,
Crawford et al., 1978; Ishizaki et al., 1978) have described fewer
than the four major nonglycosylated EIAV proteins reported by us.
Thus we sought to establish conclusively whether the four virion
proteins (p26, ppl5, pll and p9) resolved by analytical procedures
used here are unique or share amino acid sequences, perhaps as a
cleavage product of each other.
Homogeneous preparations of
3
[ H]-leucine labeled p26, ppl5, pll and p9 were isolated by a
3
preparative GHCI-GF of [ H]-leucine EIAV.
Purified proteins were
then subjected to limited proteolysis by chymotrypsin or S^. aureus
protease V8, and the resulting peptides were analyzed by SDS-PAGE, as
described by Cleveland et al. (1977).
The autoradiograph in Figure
III-5 shows that the fragments produced by protease V8 treatment of
p26, ppl5 and pll are unique.
A large amount of substrate protein in
each case remains undigested, even after 2 h of incubation.
In
contrast, p9 appeared to be digested rapidly to small fragments within
the normal 30 min digestion period, and the resulting fragments
apparently migrated from the gel.
In a complementary digestion,
carried out with chymotrypsin, SDS-PAGE analysis revealed novel
chymotryptic peptides generated from p26, ppl5 and p9; pll was
apparently cleaved to small fragments which migrated from the gel.
The unique peptides generated by the two proteases and the
characteristic efficiencies of cleavage under identical conditions
indicate that EIAV p26, ppl5, pll and p9 contain unique amino acid
sequences and therefore are not related.
b).
Two-Dimensional Peptide Mapping Procedure.
In the course of further investigation, the two-dimensional
peptide mapping procedure of Elder et al. (1977a) was developed and
used to compare EIAV proteins.
This method is more sensitive and can
easily detect different amino acid sequences between two or more
proteins.
EIAV nonglycosylated proteins were separated on SDS-PAGE by
electrophoretic analysis of about 100 ug of EIAV proteins on a slab
polyacrylamide gel.
Glycosylated proteins, gp90 and gp45, were
obtained after a lectin affinity chromatography separation, followed
69
p26
pp15
pll
p9
p26
) «*»
pp15
p11
P9
p26 pp15
p11 p9'
<*•
4 )
S ubstrate
Proteins
j
V-8 Protease Peptides
L
chym otryptic
P eptides
Figure III-5:
SDS-PAGE analysis of peptide fragments generated by
3
limited proteolysis of [ H]-leucine labeled EIAV p26, ppl5, pll,
and p9, using either S_. aureus protease V8 or chymotrypsin.
Untreated radioactive proteins are included for reference.
70
by SDS-PAGE analysis of lectin binding fraction (see below).
The EIAV
proteins, thus separated into individual components by SDS-PAGE, were
detected by Coomasie blue staining and excised from the gel.
The
coomasie blue stained gel band of each protein was radioiodinated and
digested with trypsin in situ as described (Elder et al., 1977b).
Radioiodinated tryptic peptides of each protein were then separated on
cellulose coated thin layer plates using electrophoresis in one
dimension and chromatography in the second dimension as described in
Chapter II.
Autoradiographs of such maps are presented in Figure
III-6.
The peptide maps of p26, ppl5 and p9 are unique and
characteristic of individual polypeptides (Figure III-6). The p26 map
revealed about 12 major peptides distributed in a characteristic
pattern.
The ppl5 map also demonstrated about 12 major spots, but
their distribution was distinct from that of p26.
showed only four to five iodinated fragments.
The p9 digest
None of these maps
showed common peptides as determined by their position on
two-dimensional maps.
The pll could not be radioiodinated by the
chloramine-T method used, due to lack of tyrosine residues (see below;
Montelaro et al., 1982), and hence could not be mapped by this
procedure.
Thus, the results of this more sensitive procedure
confirmed the uniqueness of these polypeptides.
Their uniqueness was
further reflected in serological differences and absence of
immunological cross-reactivity as determined by enzyme-linked
immunosorbent assays (Montelaro et al., 1982).
Peptide maps of virion glycoproteins, gp90 and gp45, are also
presented in Figure III-6.
Both of these maps are complex with more
71
Figure III-6:
Two-dimensional peptide analysis of
125
I-labeled EIAV
major proteins digested with DPCC-treated trypsin.
electrophoresis, C = chromatography.
E =
72
>-T
than 30
125
I-labeled peptides.
Such complexities may result in part
from inherent heterogeneity in these glycoproteins (Strand and August,
1977; Schultz and Oroszlan, 1979).
Also a peptide with mono and
di-iodinated tyrosine residues may migrate differently, thus
contributing further to the complexity of the map.
Regardless of the
map complexity, they are reproducible and characteristic of a
particular glycoprotein.
Maps of gp90 and gp45 clearly demonstrate
that these peptide fingerprints are distinct, and thus gp90 and gp45
are not related or do not have common amino acid sequences.
This is
in contrast to murine viruses, where gp45 has been shown to be a
degradation product of the major surface glycoprotein by peptide
mapping analysis (Elder et al., 1977a).
When analyzed on SDS-PAGE containing 7.5% acrylamide, gp45 could
be resolved into two bands.
Separate peptide mapping analysis of two
resolvable bands of gp45 gave identical maps (data not shown).
Isoelectric Focussing:
pi Determination
The isoelectric point of each major nonglycosylated polypeptide
3
was determined by subjecting purified [ H]-leucine labelled
preparations of p26, ppl5, pll and p9 to isoelectric focussing on
cylinderical polyacrylamide gels, as described earlier.
Each protein
preparation displays a single homogeneous peak of radioactivity upon
analysis by SDS-PAGE (Figure III-3C-F, Parekh et al., 1980).
The radioactivity profiles of isoelectric focussing gels in Figure
III-7 show single homogeneous peaks of radioactivity for EIAV p26
(Figure III-7A) with a pi of 6.2, and for EIAV p9 (Figure III-7C) with
a pi of 5.0.
In contrast to the single isoelectric species described
above, EIAV ppl5 reproducibly focussed in a characteristic
74
fO.O
p26
1.1
rpis
y.%
•o
o.o
o
K
40
I
O
X
to
40
00
to
40
•0
•0
SO
40
•0
•0
Diitonct MiQrottd (mm)
Figure III-7:
3
Isoelectric focussing of [ H]-leucine labeled EIAV
proteins on cylinderical polyacrylamide gels.
75
heterogeneous pattern, with major portions of the protein appearing at
pi values of 5.7, 6.6, 7.6 and 8.3 (Figure III-7B). This
heterogeneous pattern varied only slightly when ppl5 was isolated from
different virus preparations.
The phosphoproteins of avian and
mammalian oncornaviruses have been shown to display a similar charge
heterogeneity, reflecting different degrees of phosphorylation (Hayman
et al., 1977).
Subsequent results from amino acid analysis after
short term acid hydrolysis of ppl5 demonstrated that ppl5 is indeed a
phosphoprotein (Montelaro et al., 1982).
EIAV pll could not be focused on the usual gel ranging in pH from
2 to 10.
When isoelectric focussing gels were intermittantly
terminated before completion, a small peak of radioactivity was
observed migrating towards the cathode, and eventually migrating from
the gel, indicating a pi of > 10.0 (data not shown).
Phosphoprotein Analysis
One or more polypeptides of retroviruses are usually
phosphorylated (Hayman et al., 1977).
The major viral phosphoprotein
has been implicated in structural and regulatory functions (Montelaro,
et al., 1978; Leis et al., 1978) by virtue of its location in the
intact virion and its specific affinity to viral RNA.
Thus we sought
to detect possible EIAV phosphoprotein(s) by propagating
fetal equine kidney cells (Pal et al., 1975).
32
32
P-EIAV in
P-EIAV was purified
3
and subjected to SDS-PAGE analysis along with [ H]-leucine labeled
EIAV.
The resulting electrophoretic radioactivity profile is
presented in Figure III-8A.
A
32
P peak observed at the top of the gel
(4-5 mm) apparently represents high molecular weight viral genomic RNA
Figure III-8:
Phosphoprotein analysis.
(A) SDS-PAGE of 32P-EIAV
(---- ) vs[3H]-leucine EIAV (----- ) (B) GHCI-GF of 32P-EIAV
(---- ) vs
[3H]-leucine EIAV (----).
77
-3
pp15
2-
CO
gp90 p70
gp45
ip11 A
p3Q
■v._.
200
DISTANCE
ui
MIGRATED (mm)
p26
4
3
ppIS
2
1
■zre.
200
FRACTION NO.
78
which barely enters the 10% acrylamide gel.
32
In addition, the
P-radioactivity profile shows three more peaks.
A small peak at
about 55 mm coincides with p61 (See Figure III-lA) and another one at
155 mm coincides with EIAV ppl5.
A large, broad
ahead of the dye represents virion phospholipid.
32
P-peak migrating
The results seemed
to indicate that EIAV ppl5 and p61 are phosphorylated.
However, the
results of further experiments did not support this conclusion.
Samples of
32
3
P-EIAV and [ H]-leucine-EIAV were simultaneously
analyzed by GHCI-GF analysis, and the radioactivity elution profile of
such an analysis is presented in Figure III-8B.
demonstrate that a large amount of
32
The results
P label elutes in the void
volume, representing genomic viral RNA.
However, no
32
P radioactivity
3
eluted along with H-leucine labeled ppl5 or other EIAV proteins.
32
P labeled lipid eluted in the excluded volume of the column.
the
32
The
Thus,
P radioactivity peak migrating in place of ppl5 on SDS-PAGE
apparently did not represent ppl5.
That this peak is not ppl5, but
represents probably host tRNAs carried by virions, was confirmed by
its susceptibility to RNase digestion.
treated
32
P-EIAV did not show any of the
in Figure III-8A, except the
shown).
32
SDS-PAGE analysis of RNase
32
P-radioactivity peaks seen
P-labeled phospholipid peak (data not
Also, when viral RNA from
32
P-EIAV was extracted using phenol
and chloroform to remove proteins, and analyzed on SDS-PAGE,
demonstrated RNase susceptible
32
P-radioactivity peaks.
Results of amino acid analysis of ppl5 (Montelaro et al., 1982)
did provide evidence that ppl5 is, in fact, a phosphoprotein.
However, we could not incorporate
32
P in EIAV ppl5 by labeling the
propagating virus in tissue culture by the method of Pal and
79
Roy-Burman (1975).
Modifications (overnight growth in phosphate-free
media or use of undialyzed fetal calf serum) of the labeling procedure
yielded the same results.
32
The reasons for our failure to incorporate
P into EIAV ppl5 are not clear, but this may be due to suboptimal
labeling conditions for the equine kidney cells.
Radioiodination of EIAV proteins in vitro
Proteins can be radioiodinated to a very high specific activity
by several procedures (Hunter and Greenwood, 1962; McFarlane, 1958;
Bolton and Hunter, 1973; Morrison et al., 1971).
Of these,
radioiodination of tyrosine residues of protein using chloramine-T as
an oxidizing agent is a widely used method (Montelaro and Bolognesi,
1979).
125
I-labeled proteins can be used for a variety of biochemical
and immunological techniques (Mosser et al., 1975; Morrison et al.,
1971).
The major nonglycosylated polypeptides of EIAV were purified to
homogeneity by GHCI-GF analysis and subsequent ion exchange
chromatography, where required (EIAV pll and p9; Montelaro et al.,
1982).. Following iodination, TCA precipitable counts obtained for
each of these proteins were calculated for nmol amounts and these
iodination efficiencies are presented in Table III-2.
It is evident
that pll can not be labeled by chloramine-T radioiodination procedure.
This result correlates with the results of amino acid analysis of pll
(Montelaro et al., 1982), showing that pll does not contain tyrosine
residues.
Relative iodination efficiencies of p26, ppl5 and p9
perhaps reflects the accessible tyrosine residues.
80
Table III-2.
Iodination efficiencies of EIAV nonglycosylated
polypeptides by chloramine-T method.
No. of
tyrosine/molecule*
^ ^ 1 dpm/nmol
p26
4
1 x 107
PP15
3
2.3 x 106
pll
0
p9
3
* Montelaro et al. (1982).
—
6 x 107
81
III.
POLYPEPTIDE LOCALIZATION
According to the current model for oncornavirus structure, the
location of the structural polypeptides of virions can be correlated
with certain biochemical properties (Bolognesi et al., 1978; Montelaro
and Bolognesi, 1978; Montelaro et al., 1978; Stephenson et al., 1977).
EIAV is found to have a polypeptide composition analogous to that of
oncornaviruses.
This analogy included the number of major structural
polypeptides, their molecular weight range, secondary modifications,
eg. glycosylation and phosphorylation, of some polypeptides, and pi
values for major non-glycosylated proteins.
Thus, certain experiments
were performed to localize the structural components of EIAV and to
compare these localizations with those reported for prototype-C
oncornaviruses.
1.
Outer Envelope Proteins
The protease bromelain has been used routinely to distinguish
surface proteins from proteins sequestered from enzymatic digestion by
the virion lipid bilayer (Rifkin and Compans, 1971; Mosser et al.,
1975).
3
Thus, [ H]-leucine labeled EIAV was digested with bromelain, and
the treated particle was analyzed by SDS-PAGE (Figure III-9). A
14
comparative electrophoretic pattern of untreated [ C]-leucine EIAV is
shown superimposed in the same figure (broken line) as a reference.
3
The treatment of [ H]-leucine EIAV with bromelain resulted in complete
removal of the two viral glycoproteins (gp90 and gp45), whereas the
major nonglycosylated proteins (p26, ppl5, pll and p9) were not
affected by enzyme digestion.
Although, ppl5 and pll were not
resolved completely (Figure III-9), probably because of the
S D S -P A G E OF B R O M E L A IN -T R E A T E D
3 H -L e u ~ E IA V
lO
8
CPM*
CPM
x 10
12
4
bp45
5^7 w y v
0
50
100
DISTANCE
Figure III-9:
150
MIGRATED
200
250
(m m )
3
SDS-PAGE analysis of bromelain treated [ H]-leucine
14
EIAV (----) and of untreated [ C]-leucine EIAV (--- ) used as
reference.
83
comigration of some glycoprotein fragments in that region of the gel
(Montelaro et al., 1978), the presence of pll, in addition to p26,
ppl5 and p9, could be demonstrated by GHCI-GF analysis of bromelain
treated virus preparations (data not shown).
In addition, bromelain
3
digestion of [ H]-glucosamine labeled EIAV, in which only gp90 and
gp45 were radiolabeled (Parekh et al., 1980), resulted in the complete
loss of radioactivity from these virus particles.
Thus, gp90 and gp45
appear to form the surface projections of the virus, whereas p26,
ppl5, pll and p9 constitute the internal virion components.
Additional evidence for this localization came from
surface-specific radioiodination.
Lactoperoxidase catalyzed
iodination is reputed to label only polypeptides which are exposed on
the outside surface of a membrane (Marchalonis et al., 1971; Phillips
and Morrison, 1971; Rifkin and Compans, 1971; Mosser et al., 1975).^
EIAV was labeled in vitro with
125
I using lactoperoxidase, purified
and subsequently analyzed by SDS-PAGE.
The radioactivity profile
(Figure 111-10) reveals five peaks of radioactivity, two of these
coinciding with virion gp90 and gp45.
Viral proteins have not been
observed comigrating in the position of the other two peaks [between
gp90 and gp45 (MW = 68,000) and between p26 and ppl5 (MW = 20,000).
These proteins, therefore, were believed to be contaminating
proteins from the tissue culture system which copurify with virions.
The major nonglycosylated viral proteins, p26, pll and p9 were not
significantly labeled, except ppl5, which was labeled to a small
extent.
This may indicate its location close to the envelope.
Thus,
these results indicate that the two glycoproteins, gp90 and gp45,
S D S -P A G E
OF
LA CTO PER O X ID A SE-R A D IO IO D IN A TED
EIAV
16
gp 90
I-CPM x 10
I2
gp45
8
4
Ppl5
0
50
100
D IST A N C E
Figure 111-10:
EIAV.
150
200
250
M IGRATED (m m )
SDS-PAGE analysis of lactoperoxidase-radioiodinated
85
are on the outside of the lipid envelope while nonglycosylated
polypeptides p26, ppl5, pll and p9 are internal.
The envelope proteins in certain avian and murine oncornaviruses
have been observed to be intermolecularly linked by disulfide linkages
(Montelaro et al., 1978).
Thus, we sought to investigate the presence
or absence of disulfide linkages between EIAV gp90 and gp45.
For
3
these purposes [ H]-glucosamine labeled EIAV was subjected to
nonreducing SDS-PAGE analysis as described (Montelaro et al., 1978).
Analysis of the radioactivity in the gel gave a profile identical to
3
the reducing SDS-PAGE analysis of [ H]-glucosamine labeled EIAV
(Figure III-1B), demonstrating that gp90 and gp45 are not disulfide
linked.
2.
Ribonucleoprotein (RNP) Analysis
The identity of the internal protein(s) associated with the viral
RNA was determined by treating purified EIAV with Nonidet P-40 (NP-40)
to release the virion ribonucleoprotein complex, which was then
isolated by centrifugation on a sucrose-D20 gradient (Davis and
Rueckert, 1972; Mosser et al., 1975). The experiment was carried out
3
with [ H]-leucine labeled EIAV in a parallel analysis with unlabeled
3
EIAV. The analysis of NP-40 treated [ H]-leucine-EIAV on sucrose-D20
gradient is shown in Figure III-l1A.
A small peak of radioactivity
with a density of about 1.31 g/ml is observed in fractions 7 to 9.
Identical fractions from a sucrose-D20 gradient analysis of NP-40
treated unlabeled EIAV were pooled, and the proteins in the pool were
TCA precipitated and subsequently analyzed on SDS-PAGE.
The results
of SDS-PAGE analysis of proteins contained in the subparticle and
those solubilized by the detergent are presented in Figure III-lIB.
Figure III-l1:
RNP preparation and analysis.
(A) Sucrose-D20
3
gradient analysis of RNP prepared from [ H]-leucine EIAV.
(B) SDS-PAGE analysis of RNP fractions and soluble fractions
obtained from unlabeled EIAV and stained with coomassie blue.
Untreated EIAV is included as a reference.
87
FRACTION
NO
88
The ribonucleoprotein subparticle contains all of the virion pll, some
of the EIAV ppl5, p26 and a few high molecular weight components (p30,
p61 and p70), while the solubilized proteins consist of gp90, gp45,
p26, ppl5 and p9, but no pll.
The total absence of pll in the detergent solubilized portion and
its presence in subparticle fractions indicated it to be the major
component of the ribonucleoprotein complex.
Some amount of ppl5 and
p26, observed in the ribonucleoprotein fractions, may reflect their
specific association with RNA or pll or may result from incomplete
dissociation by the detergent.
The fact that pll is a basic protein
favors the idea that it is associated with negatively charged RNA
genome.
The ppl5 is a phosphoprotein and, as in other retroviruses
(Sen and Todaro, 1977), may have a specific affinity for RNA.
However, most of the ppl5 and p26 are solubilized by the detergent,
and hence their predominant location cannot be in the
ribonucleoprotein.
Thus, the results obtained here are similar to
those observed with ribonucleoprotein particles isolated from avian
(Bolognesi, 1974; Davis and Rueckert, 1972) and murine (Bolognesi,
1974) oncornaviruses, in which the basic internal protein is tightly
bound to the viral RNA genome.
The presence of several high molecular components (MW = 30,000 to
70,000) which purified with the RNP, but were absent from the
detergent-solubilized portion, suggests their exclusive location in
the RNP and may indicate a role as components of the virion reverse
transcriptase or as uncleaved polyprotein precursors of the internal
proteins (Montelaro and Bolognesi, 1978).
These minor proteins were
3
detected previously in [ H]-leucine EIAV preparations (Figure III-1A,
Table III-l), but their significance was not known.
3.
Inner Coat
In avian and murine oncornaviruses, the virion phosphoprotein is
believed to comprise the inner coat structure underneath the lipid
envelope (Montelaro and Bolognesi, 1980).
In EIAV, a small amount of
ppl5 always elutes with the lectin binding fractions when glycosylated
and non-glycosylated proteins are separated on lectin affinity column
(see below), although ppl5 is not glycosylated.
This may reflect its
affinity for envelope components such as gp45 and may also suggest its
location just underneath the envelope in the inner coat.
However,
this does not exclude its location elsewhere in the virion.
4.
Model of EIAV
\
From these localization studies, a model of EIAV can be proposed
as shown in Figure 111-12.
Knob and spike structures of the virion
are believed to be made of two glycoproteins, gp90 and gp45, which are
known to be present on the outside of the lipid envelope.
Like avian
gp35 and murine pl5E, the poorly glycosylated hydrophobic protein,
gp45, may be a transmembrane protein spanning the bilayer.
Underneath
the lipid bilayer is the inner coat, probably made of phosphoprotein,
ppl5.
The icosahedral core structure of the virion contains p26, the
major virion structural protein, and perhaps also p9.
Within the core
shell is the helical viral RNA genome with pll and reverse
transcriptase molecules closely associated to constitute the
ribonucleoprotein complex.
Figure 111-12:
A tentative model of EIAV proposed on the basis of
localization experiments.
RT = reverse transciptase.
91
Thus, the properties of constituent polypeptides and limited
studies of the localization have permitted a tentative model of EIAV
which is analogous to model of type-C retroviruses (Montelaro and
Bolognesi, 1980).
IV.
A SEARCH FOR ANTIGENIC VARIATION IN EIAV
EIAV Isolates
EIAV isolates were recovered from experimentally inoculated
ponies in which the Wyoming (parental) strain of EIAV was serially
passaged (Orrego et al., 1982).
Sources of these isolates are
demonstrated in Figure II-l showing isolate designation, inoculation/
replication path and the passage number.
Second passage and third
passage isolates (P2-1, P2-6 and P3-1, P3-3, respectively) were
recovered from chronically 111 ponies demonstrating periodic episodes
of disease.
Daily rectal temperature plots of these ponies (Orrego,
1983) are shown in Figure 111-13, in which panel A represents
temperature profile of a second passage pony.
Two isolates, P2-1 and
P2-6, used in this investigation, were recovered from plasma samples
of this pony during the first and sixth fever episodes, respectively,
as shown.
Panel B contains a temperature plot of a third passage pony
from which EIAV isolates P3-1 and P3-3 were recovered.
The results of
Orrego (1983) demonstrated that these isolates are distinct in
neutralization assays, in agreement with the data of Kono et al.
(1971, 1973), and hence are good candidates for the study of antigenic
variation.
For the biochemical work presented here, viruses recovered
from endpoint dilutions of plasma collected were propagated in fetal
equine kidney cells and purified by standard procedures.
92
P2-6
pji
40
38;
90
60
120
DAYS
PM
150
180
210
240
270
POST-INOCULATION
B
P
a;
5
IXJ
30
P2-1
Figure 111-13:
DAYS
60
90
120
ISO
POST-INOCULATION
Rectal temperature plots of (A) second passage and
(B) third passage ponies undergoing febrile episodes (Orrego,
1983).
Viral inoculum and recovered virus isolates used in this
investigation are shown.
93
Comparison of EIAV Isolates
Antigenic variation can be studied by analyses of the component
proteins by SDS-PAGE (Kew et al., 1981; Nottay et al., 1981) and by
peptide mapping procedures (Scott et al., 1979; Kew et al., 1980).
Both of these techniques are used here to compare the major antigens
of EIAV isolates.
(a)
Analysis by SDS-PAGE
The polypeptide composition of the six virus strains was
initially compared by SDS-PAGE analysis.
The data presented in Figure
111-14 demonstrates no major differences among the polypeptides found
in parental and other EIAV isolates; the apparent molecular weights
and relative stoichiometry of the major virion proteins p26, ppl5, pll
and p9 are indistinguishable.
Because of the complex protein profile
in the region of the gel containing gp90 and gp45, it was difficult to
compare these components from SDS-PAGE of whole virus preparations.
Thus, the virion glycoproteins and nonglycosylated proteins were
separated on Sepharose bound Lens culinaris (lentil) lectin.
As
reported in detail previously (Montelaro et al., 1983), the affinity
chromatography fractionation of EIAV accomplishes the efficient
separation of nonglycosylated and glycosylated virion components.
Glycosylated virion components (binding fractions) of EIAV isolates
along with that of parental strain are analyzed by SDS-PAGE (Figure
111-15).
The glycoprotein fractions contain the virion gp90 and gp45,
a few other nonviral glycoproteins, and a small amount of EIAV ppl5.
In contrast, the nonbinding fraction contains essentially all of the
virion p26, ppl5, pll and p9 components and a number of other minor
virion associated proteins (data not shown).
Figure 111-14:
SDS-PAGE analysis of proteins of EIAV isolates stained
with cooraassie blue.
Parental EIAV is included for comparison.
# ppl5
Figure 111-15:
SDS-PAGE analysis of glycoprotein pools of EIAV
isolates, stained with coomassie blue.
Parental EIAV
glycoprotein pool is included for comparison.
96
The following observation can be made from the results shown in Figure
111-15:
i) Variable amounts of glycoproteins are recovered from
different isolates, starting with approximately the same amounts of
viral proteins (about 2 mg in each case). This may reflect differing
solubility of viral glycoproteins present in these viruses,
ii) The
respective glycoproteins, especially gp90, demonstrated different
migration rates and differences in band width, suggesting structural
alterations,
iii) The gp45 band, in most cases, is very faint,
migrating as a doublet (see parental EIAV) in 7.5% acrylamide gels.
The generally low recovery of gp45 may be due to its low affinity for
lectins and its hydrophobic, insoluble nature.
Analysis by Two-Dimensional Peptide Mapping
Although the SDS-PAGE analysis of EIAV proteins indicated some
structural variation in glycoproteins, a more definitive and
informative approach is necessary to detect structural alterations
related to antigenic variation in EIAV.
From the SDS-PAGE analysis of
whole virions (Figure III-I4) and their respective glycoprotein pools
(Figure 111-15), protein bands corresponding to gp90, gp45 and p26
were excised from all the EIAV isolates, radioiodinated and digested
with trypsin in situ, and the resultant radioactive tryptic peptides
analyzed by two dimensional mapping procedures, according to the
methods of Elder et al. (1977a).
In some cases, EIAV ppl5 and p9 were
also mapped by the above procedure.
The comparative analysis carried
out here (Figure 111-16, 111-17, and 111-19) represent a pairwise
study of the virus isolates involving a virus isolate and the
preceding virus strain from which it originated, and these pairs are
identified by arrows in the figures.
97
Analyses of gp90
Comparative peptide maps of the gp90 components of EIAV isolates
are presented in Figure 111-16 in which the parental EIAV gp90 map is
also included for reference.
Examination of the gp90 peptide maps
reveals that PI-1 gp90 is very similar to parental gp90.
Prominent
peptides migrate at the same position on two-dimensional maps with
only a few differences.
Two peptides are deleted (open circles) and
two are labeled very poorly (broken circles) when Pl-1 gp90 map is
compared to Parental EIAV gp90 map.
Differences between the maps also
include 2 peptides which are new to Pl-1.
The gp90 molecule showed many differences when Pl-1 was
inoculated in a second passage pony to generate P2-1. These include 6
new peptides not seen previously (Pl-1 gp90 map), two deletions and
two sites where peptides seem to have rearranged (compare P2-1 map to
Pl-1 map).
The replication of P2-1 in the second passage pony produce the
P2-6 isolate.
The P2-6 gp90 map showed deletion of two peptides and
only one new peptide, when compared to the P2-1 gp90 map.
The P3-1 gp90 was different from the P2-1 gp90 molecule as seen
from their peptide maps where P3-1 gp90 showed four deletions, one
poorly labeled peptide and one area of peptide rearrangement.
The
P3-1 gp90 map had a high background, and as a result certain areas
indicated with a question mark could not be compared.
In contrast,
the P3-3 gp90 gave a clean map demonstrating 3 new peptides, one
deletion and one very poorly labelled peptide relative to its
predecessor P3-1.
A clean gp90 map from P3-3 is perhaps a reflection
98
Figure 11-16:
125
Comparative two-dimensional peptide map analysis of
I-labeled gp90 digests of EIAV isolates.
map is included as a reference.
Parental EIAV gp90
(O) missing peptides, (CO
peptides with, decreased labeling, (►) new peptides and (-*)
rearranged peptides.
E = electrophoresis, C = chromatography.
99
I
parental
EIAV
P2- 6
P3-3
100
of the fact that the gp90 molecule from this isolate gave a
comparatively tight band on SDS-PAGE, showing less heterogeneity.
From the results shown here it seems there are alterations in
glycoprotein structure associated with EIAV antigenic variation.
Moreover, there appears to be more alterations in the gp90 molecule
when virus was adapted to another animal than when allowed to
replicate in a single animal.
Analyses of gp45
Comparative peptide maps of the gp45 components of the EIAV
isolates are presented in Figure 111-17.
When the Pl-1 gp45 is compared to parental EIAV gp45, one new
peptide is evident while another one is labeled very poorly.
also one area of peptide rearrangement.
There is
However, the Pl-1 gp45 map
had a high background in some areas, and, consequently comparisons
cannot be termed definitive.
Relative to Pl-1, the P2-1 gp45 gave a
peptide distribution showing two new peptides, one probable deletion
and one area of peptide rearrangement.
Here also there was one
questionable area due to high background.
In going from P2-1 to P2-6,
the gp45 gave a map with one peptide showing altered mobility and one
probable deletion.
Compared to P2-6, the P3-1 gp45 showed two
additional peptides, one deletion and two peptides which labeled
poorly, in addition to two areas of some altered distribution as
marked.
In contrast P3-3 gave a map showing only 3 additions compared
to the P3-1 gp45 map; all other peptides were identical.
It was noted that the recovery of gp45 from the virus isolates
was very poor and this may have contributed to the high backgrounds of
certain two-dimensional maps, making reliable comparisons difficult.
101
Figure 111-17:
125
Comparative two-dimensional peptide map analysis of
I-labeled gp45 tryptic digests of EIAV isolates.
EIAV gp45 map is included as a reference.
Parental
(O) missing peptides,
(O) peptides with decreased labeling, (►) new peptides and (-»)
rearranged peptides.
E = electrophoresis, C = chromatography.
102
P arental
E IA V
103
In any case, alterations appear to occur among the gp45 molecules from
various virus strains and this variation may be important with regards
to antigenic variations in EIAV.
Glycopeptides
The results presented above demonstrate that EIAV glycoproteins
change during virus replication in an animal but give no information
whether the changes involve glycosylated or nonglycosylated regions of
the polypeptide.
For this purpose, it is important to know which of
the peptides in the gp90 map are glycosylated and which are
nonglycosylated.
Thus, an
125
I-labeled tryptic digest of the P2-6
gp90 was separated into glycosylated and nonglycosylated pools by
affinity chromatography on lentil lectin as described (Montelaro et
al., 1982), and the binding and nonbinding peptide pools were analyzed
by two dimensional peptide mapping procedures.
The nonglycosylated
peptides produce a map (Figure III-18A) representing a partial gp90
map (compare Figure 111-16, P2-6 map).
However, when the glycosylated
peptides are analyzed by the same procedure, it was found that the
radioiodinated peptides of gp90 were resolved only poorly during
electrophoresis, and failed completely to chromatograph in the second
dimension (Figure III-18B). This observation was confirmed by peptide
3
analysis of a digest of purified [ H]-glucosamine labeled gp90 (Parekh
et al., 1980) (Figure III-18C).
This poor resolution precluded any
comparison of glycopeptides by this mapping procedures.
It also
indicated that the differences observed in the maps of radioiodinated
gp90 probably do not involve glycosylated peptides.
Thus, further
structural alterations may be detectable upon analysis of
A
B
Figure 111-18:
(A)
(B)
125
125
C
Two-dimensional peptide analysis of gp90 digest.
I-labeled lectin nonbinding (nonglycosylated) peptides.
I-labeled lectin binding (glycosylated) peptides.
3
(C) [ H]-glucosamine labeled gp90 digest.
105
glycopeptides by gel filtration chromatography and/or HPLC techniques
(Hunt et al., 1981).
Analyses of Internal Antigens
The major internal protein, p26, recovered from SDS-PAGE analysis
of these virus isolates was mapped and the results are shown in Figure
111-19.
Parental EIAV p26 and Pl-1 p26 gave identical maps; no
detectable changes in the tryosine containing
are evident.
125
I-labeled peptides
When the Pl-1 p26 map is compared to P2-1 p26, all of
the major peptides are found to be identical, except one in P2-1 which
was not apparent in the Pl-1 p26 map.
When p26 molecules from the
P2-1 and P2-6 are compared, they gave very reproducible maps
demonstrating no changes in this molecule during replication in the
pony.
However, comparison of p26 from P2-1 and P3-1 did indicate some
changes in the molecule.
Thus this major internal protein seems to
have undergone structural alteration when the virus was transferred
from one animal to another.
In contrast, P3-3 produced a p26 map
identical to the P3-1 p26, suggesting that this molecule is conserved
while the virus is replicated in a single animal.
In addition to p26, P2-1 p9 was mapped by identical procedures
and found to have peptide maps identical to the analogous parental
antigen (Figure 111-21).
Also, P2-1 ppl5, P3-1 ppl5 and P3-3 ppl5
were mapped to see if the observed alteration in p26 extended to other
internal antigens.
This molecule from all three isolates gave maps
identical to parental ppl5 (Figure 111-20).
These results indicate
that ppl5 and perhaps p9 do not change during backpassaging of the
virus or replication in a single pony.
106
Figure 111-19:
125
Comparative two-dimensional peptide analysis of
I-labeled p26 tryptic digests of EIAV isolates.
p26 map is included as a reference.
(-*) rearranged peptides.
chromatography.
Parental EIAV
(►) new peptides and
E = electrophoresis, C =
107
Figure 111-20:
125
Comparative two-dimensional peptide analysis of
I-labeled ppl5 tryptic digests of some EIAV isolates.
Parental EIAV ppl5 map is included as a reference.
109
P a ren tal E I A V
P2-1
*•**
%
Parental EIAV
Figure 111-21:
Two-dimensional peptide analysis of
trypitc digests of parental EIAV and P2-1.
125
I-labeled p9
CHAPTER IV
DISCUSSION
The experiments described in this investigation have enabled us
to achieve the following goals.
a)
Identification of the major and minor structural polypeptides of
EIAV.
b)
Characterization of the major polypeptides with respect to their
secondary modifications (e.g. glycosylation, phosphorylation),
isoelectric points, peptide analyses and a few other characteristic
properties.
c)
Assignment of the major polypeptides to specific locations in the
intact virion and the proposal of a tentative model for EIAV.
d)
An analysis of the molecular nature of antigenic variation in EIAV
by peptide mapping of antigens of EIAV isolates recovered from febrile
episodes in experimentally inoculated ponies.
Previous workers typically used only one method for EIAV
polypeptide analysis and molecular weight estimation.
In contrast,
the complementary techniques of guanidine hydrochloride gel filtration
and high resolution SDS-PAGE have been used in this investigation to
resolve EIAV structural polypeptides.
This was very useful since low
molecular weight polypeptides, especially ppl5 and pll, resolved
better on GHCl-GF than on SDS-PAGE.
Also, the results obtained by
these two techniques were used for molecular weight estimations and
nomenclature as recommended by August et al. (1974).
Our experiments
demonstrated that EIAV grown in fetal equine kidney cells contains six
111
112
major polypeptides and four minor components.
The major polypeptides
included 2 glycoproteins and four nonglycosylated polypeptides.
Cheevers et al. (1978) reported the presence of four
glycoproteins gp77/79, gp64., gp40 and gplO, the first three of which
were major structural components.
In contrast, we demonstrate that
EIAV contains only two glycoproteins, gp90 and gp45, which together
represented less than 10% of the virion proteins.
Ishizaki et al.
(1978) concluded that EIAV contains two major glycoproteins,
3
designated as gp80 and gp40, while the [ H]-glucosamine labeled peak
obtained in the region of gplO was attributed to glycolipid.
It seems likely that the gp64 component reported by Cheevers et
al. (1978) most probably represented glycoprotein from tissue culture
system which copurifies with the virus. In our analysis, we found
3
that partially purified [ H]-glucosamine labeled virus preparations
contain a glycoprotein analogous to gp64 which is present in varying
amounts, but absent after further purification of the virus by
isopycnic centrifugation.
Also analysis of supernatants from
uninfected kidney cells demonstrated the presence of an analogous
component.
Thus, this glycoprotein was not considered to be an
integral component of the purified EIAV.
Cheevers et al. (1978) and Ishizaki et al. (1978) also reported
that EIAV contains two (p29 and pl3) and three (p25, pl4 and pll)
major nonglycosylated polypeptides, respectively, as analyzed by
SDS-PAGE.
Examinations of the polypeptides of visna virus, a
prototype lentivirus, similarly revealed two (Bruns and Frenzel,
1979) or three (Lin, 1977; Scott et al., 1979) major nonglycosylated
proteins.
Based on these observations it has been suggested that
113
polypeptide composition may serve as a distinguishing feature between
the lentivirus and oncornavirus subfamilies of the family Retrovividae
(Matthews, 1979).
However, we reported here that EIAV contains four
major nonglycosylated proteins designated p26, ppl5, pll and p9.
These findings are in agreement with those of Charman et al. (1979),
who resolved four structural components [p26, pl2, plO (acidic) and
plO (basic)] by isoelectric focussing.
We further confirmed that our improved resolution of major
nonglycosylated polypeptides is not the result of degradation or
cleavage of one polypeptide into another, as limited proteolytic
digestion with two different enzymes, chymotrypsin and J3. aureus V8
protease, produced unique peptides from each of these polypeptides.
Also exhaustive digestion of radioiodinated polypeptides with trypsin
yielded peptides which displayed different two dimensional
maps, characteristic of each polypeptide and demonstrating no
relatedness.
Thus p26, ppl5, pll and p9 are unique structural
components of EIAV.
That these major structural proteins are coded by the virus and
are not cellular or serum proteins incorporating into the virus
structure was deduced by the following observations.
a)
When infected cells are metabolically labeled with radioactive
leucine, even for a short term, purified EIAV demonstrated the
presence of gp90, gp45, gp26, ppl5, pll and p9.
b)
The proportion of these polypeptides in purified virus preparation
was constant, as determined by the amount of the radioisotope
incorporated.
A polypeptide fortuitously copurifying with virions
will not always be in the same proportion.
114
c)
When EIAV was grown on different cell types, e.g. fetal equine
kidney cells or equine dermal cells, purified virus always displayed
identical major polypeptides.
d)
Artifically or naturally infected horses suffering with chronic
EIA developed antibody responses to all the major antigens described
here.
Hence these proteins can not be host cell derived, but must
represent proteins coded by the viral genome.
Our results also conclusively demonstrate that EIAV, a putative
lentivirus, contains polypeptide composition analogous to type-C
viruses, like oncornaviruses, in spite of the lack of cross-reactivity
between EIAV and any other known virus belonging to the Retroviridae
(Charman et al., 1976; Dalton, et al., 1975; Stowring et al., 1979).
Other members of the Retrovirus family with similar properties, but
unique serological specificities, are bovine leukemia virus (BLV),
Mason-Pfizer monkey virus (MPMV) and mouse mammary tumor virus (MMTV)
(Charman et al., 1976).
Although the above viruses are usually not
considered C-type viruses on morphological grounds, it is known that
avian oncornaviruses, which represent true C-type viruses, are also
unrelated serologically and biochemically to mammalian oncornaviruses.
Cheevers et al. (1980) reported that only EIAV gp79 located is on
the outside of the lipid envelope, while the other two reported
glycoproteins, gp64 and gp40, are internal.
Localization studies
carried out in this investigation clearly demonstrated that both gp90
and gp45 can be labeled by surface labeling procedures and are also
susceptible to protease digestion in the intact virions, indicating
that they are on the outside of the lipid envelope.
115
Several reports have described a high degree of similarity
between gp71 and a minor component gp45 in murine leukemia virus.
Elder et al. (1977a) reported that gp45 and gp71 generated identical
tryptic peptides, and hence differ only by the lower carbohydrate
content of gp45 (Marquadt et al., 1977).
Other workers, however, have
indicated that gp45 is derived from gp71 by proteolytic cleavage,
probably at the carboxyl end (Henderson et al., 1978).
Our results
with EIAV gp90 and gp45 showed that these glycoproteins gave very
different tryptic peptide maps and, thus, are not related to each
other by common amino acid sequences.
This was in contrast to
Cheevers et al. (1980) who concluded that EIAV glycoproteins (gp79,
gp64 and gp40 as reported) are related.
However, these investigators
made no attempts to purify EIAV glycoproteins before they were mapped.
Considering the low amounts of these proteins present in the virion,
and comigrating proteins on SDS-PAGE (Montelaro et al., 1983),
complete purification Is essential.
In the case of avian viruses,
Mosser et al. (1977) demonstrated by peptide mapping analysis that
gp35 is not a cleavage product of gp85.
In this respect EIAV
resembles avian type-C viruses.
An interesting observation from our studies was that EIAV gp45
separated into two bands in SDS-PAGE, but that the two bands of gp45
gave identical maps on peptide analysis.
Qualitative/quantitative
differences in the glycosylation of the same polypeptide may result in
a doublet.
In MMTV, a type-B retrovirus, gp36 often migrates as a
doublet in SDS-PAGE, which when subjected to tryptic peptide analysis
gave identical maps (Dickson et al., 1976).
It was later shown that
116
resolvable species of gp36 differed in their carbohydrate content
(Yagi et al., 1978).
Compared to EIAV gp90 and gp45, analogous surface components in
avian type-C viruses are gp85 and gp35 while in murine viruses they
are gp71 and pl5E/pl2E.
Both avian gp35 and murine pl5E molecules are
hydrophobic like EIAV gp45 and require detergents to keep them in
solution.
Thus, hydrophobic gp45 may span the lipid bilayer, while
the heavily glycosylated gp90 may be more exposed to the outside.
However, unlike avian viruses where greater than 90% of gp85 and gp35
are disulfide linked into a viral glycoprotein complex (Leamnson and
Halpern, 1976; Montelaro, et al., 1978), EIAV glycoproteins are not
disulfide linked.
In Murine virus FLV, only a small fraction of gp71
molecules is disulfide linked to pl5E in a complex (Montelaro et al.,
1978).
Several workers have reported the major group specific antigen of
EIAV as a polypeptide with a molecular weight of 25,000 - 29,000
daltons (Barta and Issel, 1978; Cheevers et al., 1978; Ishizaki et
al., 1978).
Here it has been termed as p26.
EIAV p26 appears to be
the major internal component analogous to avian virus p27 and murine
virus p30 (Table IV-1), partially constituting the icosahedral core
shell that lies beneath the lipid envelope and surrounds the RNP.
EIAV p26 displayed a pi of 6.2 upon isoelectric focussing.
Analagous
p30 of various mammalian type-C viruses displayed pi values ranging
from 6.2 to 8.0 (Stephenson et al., 1977).
Murine and avian retroviruses contain basic polypeptides plO and
pl2, respectively, which are believed to be associated with genomic
RNA in a ribonucleoprotein (RHP) complex.
Our results demonstrated
117
Table IV-1:
EIAV
gp90
Analogous polypeptides of EIAV, murine type-C (FLV) and
avian type-C (RSV) viruses.
Murine
type-C Viruses*
8P71
Avian
type-C Viruses*
gp85
")
J
Location
Envelope
gp45
pl5E/pl2E
gp35
p26
p30
p27
Core shell
PP15
PP12
PP19
Inner coat
pll
plO
pl2
RNP complex
p9
pl5
pl5
Core associated
* Montelaro and Bolognesi (1980).
118
the presence of such a basic polypeptide, pll, in EIAV as part of its
RNP complex.
Davis and Rueckert (1972) presented evidence that the
fast sedimenting component isolated from NP-40 treated Rous sarcoma
virus (RSV) is RNP.
This particle contained the viral 60S to 70S RNA
and some 4S RNA (host tRNAs) in a complex with a few viral
polypeptides.
Several electron microscopy studies of RNA tumor virus
structure have presented evidence that the RNP is a coiled filament
(Bonar et al., 1964; de-The' and O'Connor, 1966; Lacour et al., 1970;
Nowinski et al., 1970).
Sarkar et al. (1971) and Bolognesi et al.
(1978) proposed a general model for RNA tumor virus ultrastructure in
which the filamentous RNP is packaged within the virus core in a
helical configuration.
The basic nature of EIAV pll and its location
in RNP suggest that the polypeptide may function in packaging of the
genomic RNA or may affect the expression of the genome during
infection.
Long et al. (1980) reported the presence of a basic polypeptide
in EIAV that binds to single-stranded calf thymus DNA and apparently
is analogous to pll reported here.
However, that was the smallest
EIAV polypeptide they resolved on SDS-PAGE, whereas our results
indicated that it is the second smallest polypeptide of EIAV.
before, p9 was not resolved by these workers.
As
This polypeptide, in
contrast, is acidic and is not a component of the RNP.
The RNP substructure isolated from EIAV contained a few other
polypeptides in addition to pll (Figure III-12B).
However, only some
of these polypeptides were enriched in RNP compared to whole virus
preparation, such as p30, p61 and p70.
These proteins, like pll, were
completely absent in the solubilized protein portion which contained
119
all of virion gp90, gp45 and most of the p26, ppl5 and p9.
Other
polypeptides in the RNP may represent genome associated reverse
transcriptase (Montelaro and Bolognesi, 1980) and/or uncleaved
polyprotein precursors.
Reverse transcriptase of retroviruses is
present in about 20-70 copies per virion (Krakower et al., 1977; Panet
et al., 1975).
Two polypeptides contained in RNP, p61 and p70, are
present in about 60-70 copies each per virion (Table III-l).
These
suggest that either one or both may represent EIAV reverse
transcriptase.
A 70,000 daltons polypeptide has been identified from
EIAV to contain reverse transcriptase activity (Archer et al., 1977;
Charman et al., 1976; Cheevers et al., 1978).
Based on the evidence of phosphorylation (Montelaro et al.,
1982), EIAV ppl5 may be the analog of avian ppl9 and murine ppl2
(Table IV-1).
Murine ppl2 was shown to be present in several
different but specific phosphorylated states (Hayman et al., 1977; Pal
et al., 1975).
EIAV ppl5 displayed a heterogeneous but reproducible
pattern on isoelectric focussing gels which may correlate with the
degrees of phosphorylation of the molecule.
Avian ppl9 and murine ppl2 are believed to comprise the inner
coat in the respective viruses (Bolognesi et al., 1978).
may also constitute the analogous inner coat structure.
EIAV ppl5
Our results
indicated that some amount of ppl5 always eluted in the binding
fractions along with viral glycoproteins when detergent disrupted EIAV
was analyzed on lectin affinity chromatography, suggesting some
affinity to glycoprotein(s). This affinity may reflect its location
close to glycoproteins underneath the lipid bilayer.
Lactoperoxidase
catalyzed surface radioiodination of the intact virus resulted in
120
major labeling of glycoproteins and minor incorporation into ppl5
(Figure 111-10).
Thus ppl5, to a small extent, is accessible to
this surface labeling technique.
All of these results collectively
support the idea that ppl5 forms the inner coat structure.
At least a few molecules of retrovirus phosphoprotein are
believed to be associated with RNA genome by highly specific
interactions (Sen and Todaro, 1977; Pal and Roy-Burman, 1975).
The
presence of a small amount of ppl5 in the RNP of EIAV indicates that
it may be a possible RNP component.
But it may also bind RNA as a
result of the detergent disruption.
EIAV p9 was found to be an acidic polypeptide with pi of 5.0.
The analogous polypeptide in avian and murine viruses is the remaining
gag gene coded internal protein, pl5 in both cases.
Murine pl5 is
hydrophobic and has a pi of 7.5, and the avian pl5 is a protease with
pi of 6.6.
The exact location of EIAV p9 is not determined, but
tentatively has been assigned to icosahedral core because of its
absence both in RNP and on the outside of the lipid envelope.
Initial biochemical characterizations of EIAV polypeptides
provided a foundation to study the process of antigenic variation in
this retrovirus.
Major attention was focussed on the surface
glycoproteins gp90 and gp45 which are believed to be the main targets
of the neutralizing antibodies, although certain nonglycosylated major
internal polypeptides were also studied.
Glycosylated proteins were
separated from nonglycosylated components by lectin affinity
chromatography as described (Montelaro et al., 1983).
The lectin
isolated from Lens culinaris, a common lentil, has a binding
specificity for a-glucopyranosyl and a-mannopyranosyl residues.
121
Binding of EIAV glycoproteins to this lectin was easily reversed by
inclusion of a-methylglucoside in the elution buffer.
Thus, lentil
lectin effectively separated EIAV glycosylated and nonglycosylated
polypeptides with recoveries of about 65%.
Similar protocols were followed for EIAV isolates recovered from
infected ponies.
Although more than 80 EIAV isolates were recovered
from infected animals (Orrego, 1983), only five of these were
selected, in addition to the parental EIAV strain, for a judicious
comparative study of possible antigenic variations presented here.
Selection of isolates was based primarily on two criteria.
1)
Study of the proposed antigenic variation within an infected
animal.
2)
Study of the antigenic variation on subsequent passaging of the
virus in different animals.
Orrego (1983) demonstrated that the isolates chosen here were
serologically distinct in virus neutralization assays and consequently
were good candidates for antigenic variation studies.
From the tryptic peptide maps of gp90, it was observed that there
were a few reproducible changes detected during replication in an
infected animal (Figure 111-16, see P2-1 and P2-6; P3-1 and P3-3).
However, more variation was observed when the virus was transferred
from one animal to another than during replication in a single animal
(Figure 111-16, compare Pl-1 and P2-1; P2-1 and P3-1).
It seems that
this adaptation to physiological environment of a different animal
results from more mutational events than that observed during
sequential febrile episodes in the infected animal.
Since all the
isolates recovered from a single pony were not studied, it is not
122
possible to suggest how many changes may actually result in the
appearance of a new isolate, but these kinds of studies will indeed be
helpful.
The results obtained upon analyses of EIAV gp45 (Figure 111-17)
indicated that this molecule also undergoes structural alterations,
thus contributing to antigenic variation.
As in the case of gp90,
more variation was observed in the gp45 molecule when the virus was
transferred from one animal to another.
However, some of the gp45
maps (Pl-1, P2-1) had high background, thus hampering a definitive
interpretation.
The main reasons for this background problem are that
only small amounts of gp45 could be recovered from EIAV isolates
(Figure 111-15) and secondly, contaminating components from acrylamide
incorporate label and contribute to the background (Elder et al.,
1977b).
Although the major internal proteins did not show alterations
when the Wyoming strain of EIAV was adapted to animals, EIAV p26
demonstrated some variation when the virus was further passaged from
one animal to another (Figure 111-19).
Thus, on second back
passaging, p26 showed one new peptide (P2-1 map) that was absent in
Pl-1.
On third back passaging, more variations in p26 were observed
(compare P3-1 to P2-1).
However, alterations were limited to
adaptation of the virus to another animal and were not seen when the
virus was replicated in a single animal for a long period.
No
accompanying variation was observed in EIAV ppl5 and p9 which yielded
reproducible peptide maps.
EIAV p26 is a major internal antigen of the virus and is not
believed to be a target for neutralizing antibodies.
Selection and
123
amplification of EIAV variants may be under immunological pressure,
but this should not account for alterations in p26 molecule.
Thus, it
would appear that random mutational events in the genome of EIAV led
to these changes which also included selective alterations in the
envelope proteins.
The exact nature of the selective pressure exerted
on viruses replicating in animals is poorly defined.
It is very
likely that factors other than host antibodies may also play a role
(Palmenberg et al., 1979; Kew et al., 1981).
In the case of
poliovirus, changes in the virus polypeptides were not restricted to
the structural proteins, but were also observed in the non-capsid
proteins functioning only intracellularly (Kew et al., 1981).
Retroviruses can evolve in response to altered selection
pressure.
This was confirmed from the observation that when cloned
ASV was repeatedly passaged in cultures of chicken cells,
oligonucleotide maps of the genomes of the virus preparations
recovered were found to be altered (Coffin et al., 1980), although the
altered region of the genome was not determined.
However, all avian
viral proteins exhibit some degree of polymorphism.
Variations are
often detected when genomes or analogous gene products of different
strains of virus are examined by techniques such as oligonucleotide
mapping, peptide mapping, radioimmunoassay and SDS-PAGE (Bhown et al.,
1980; Shaikh et al., 1978, 1979).
In the case of MMTV, a B-type
retrovirus, gp52 and p27 from different strains have been shown to
possess structural differences.
It is possible that different strains
of a virus are generated by accumulation of alterations in the genome.
Recombination with endogenous genomes can also play a role in the
generation of polymorphic forms.
124
There are no reports indicating that EIAV undergoes antigenic
variation when grown in tissue culture.
Also, we have found no
evidence that in tissue culture EIAV polypeptides are altered by
repeated passage.
The tryptic peptide maps of the major antigens of
EIAV have been found to be completely reproducible in different
preparations of the virus grown in tissue culture and remained
unaltered on repeated subpassaging.
With regards to antigenic variation in EIAV, it was realized that
the changes in peptide maps of EIAV antigens detected here are
not necessarily comprehensive, since the procedure detected only
tyrosine containing peptides.
Variations in the regions of
polypeptide lacking tyrosine may go undetected.
Also, the results
indicated that the resolved peptides are, most probably, not
glycosylated (Figure 111-18).
Thus, it is very likely that there may
be additional differences, especially in the glycosylated region of
the polypeptides.
The use of different enzymes for generation of
peptides, glycoprotein labeling with radioactive sugars and the use of
HPLC for peptide analysis should provide additional information
relative to the biochemical nature of the antigenic variation detected
in these studies.
While the analyses of EIAV polypeptides for the study of
antigenic variation promises to be informative, these results need to
be correlated at the level of viral genome.
Thus, oligonucleotide
mapping of the RNAs from different EIAV isolates can determine the
changes in the viral genome and thus, explain the basis of antigenic
variation.
125
Antigenic variation has not been reported in oncornaviruses, at
least during replication in a single animal, although genetic changes
do occur through recombination at a very high frequency resulting from
a mixed infection (Coffin, 1980).
Also the existence of different
strains of viruses and occurrence of various strains in practically
all organisms suggest that antigenic variation may be a normal process
of evolution.
The difference may be in the rate at which the
antigenic variation occurs for a particular life form.
In an animal,
the obvious selection pressure is immunological, but there may be
other less obvious ones.
In nature there may be different constraints
which exert varying degrees of pressure on different life forms to
evolve.
Antigenic variation, in a sense, seems one facet of the
natural process of evolution.
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APPENDIX 1
•LIST OF ABBREVIATIONS
AGID
Agar gel immunodiffusion
ASV
Avian sarcoma virus
BSA
Bovine serum albumin
DNP-ala
2,4-dinitrophenyl alanine
DPCC-trypsin
Diphenyl carbamyl chloride treated trypsin
DOC
7-deoxycholate
EDTA
Ethylene dinitrilo tetraacetic acid
EIA
Equine infections anemia
EIAV
Equine infections anemia virus
FLV
Friend murine leukemia virus
GHC1-GF
Guanidine hydrochloride gel filtration
HA
Hemagglut inin
Idu
Iododeoxyuridine
MMTV
Mouse mammary tumor virus
MPMV
Mason-Pfizer monkey virus
NA
Neuraminidase
NP-40
Nonidet P-40
RNP
Ribonucleoprotein complex
RSV
Rous sarcoma virus
SDS-PAGE
Sodium dodeyl sulfate-polyacrylamide gel electrophoresis
TCA
Trichloroacetic acid
Tris
Tris (hydroxymethyl) aminomethane
139
VITA
December 29 , 1951
Born, Bhavnagar, Gujarat, India.
1957-1968
Attended Home School, Bhavnagar, India.
1968-1972
Attended University of Bombay, Bombay, India.
1973
Awarded a B.Sc. in Chemistry (major) and Botany
(minor).
1973-1975
Attended University of Bombay.
1975
Granted M.Sc. in Biochemistry.
1976-1978
Research assistant at Microbiology Department,
M.S. University, Baroda, India.
1979
Admitted to Louisiana State University, Baton
Rouge, Louisiana.
1983
Candidate for Ph.D. in Biochemistry, Louisiana
State University, Baton Rouge, Louisiana
140
EXAMINATION AND THESIS REPORT
Candidate:
B h a r a t S. P ar e k h
Major Field:
B io ch em istry
1 itle of Thesis:
E q u in e I n f e c t i o u s Anemia V i r u s :
P o l y p e p t i d e s and t h e B i o c h e m i c a l
C h a r a c te r iz a tio n
of V irio n
N ature o f A n t ig e n ic
V a r ia tio n .
Approved:
/Qtocddl C rfe/CtMxAD
Major Professor and Chairman
Dean of the Graduate S
EXAMINING COMMITTEE:
VMi
Date of Examination:
6/15/83