Poliovirus receptor recognition: Visualization, kinetics, and thermodynamics
Brian M. McDermott, Jr.
Submitted in partial fulfillment of the
requirements for the degree
of Doctor of Philosophy
in the Graduate School of Arts and Sciences
COLUMBIA UNIVERSITY
2001
© 2001
Brian M. McDermott, Jr.
All Rights Reserved
Abstract
We have initiated studies to understand the interaction of poliovirus with its cellsurface receptor on a biochemical and biophysical level. This thesis contains the results
of three studies. First, the kinetics and equilibrium of the poliovirus receptor interaction
have been analyzed using surface plasmon resonance. Second, the interaction was
visualized using cryoelectron microscopy and image reconstruction. Third, the activation
energy was determined for the receptor-catalyzed transition from the native virion to the
altered particle.
To study the kinetics and equilibrium of poliovirus binding to the poliovirus
receptor, we used surface plasmon resonance to examine the interaction of a soluble form
of the receptor with poliovirus (sPvr). Soluble receptor purified from mammalian cells is
able to bind poliovirus, neutralize viral infectivity, and induce structural changes in the
virus particle. Binding studies revealed that there are two binding sites for the receptor on
the poliovirus type 1 capsid, with affinity constants at 20 °C of KD1 = 0.67 µM and
KD2 = 0.11 µM. The relative abundance of the two binding sites varies with temperature.
At 20 °C, the KD2 site constitutes approximately 46% of the total binding sites on the
sensor chip, and its relative abundance decreased with decreasing temperature such that,
at 5 °C, the relative abundance of the KD2 site is only 12% of the total binding sites.
Absolute levels of the KD1 site remained relatively constant at all temperatures tested. The
two binding sites may correspond to docking sites for domain 1 of the receptor on the
viral capsid, as predicted by a model of the poliovirus-receptor complex. Alternatively,
the binding sites may be a consequence of structural breathing, or could result from
receptor-induced conformational changes in the virus.
We have studied the poliovirus-receptor interaction by using cryoelectron
microscopy to determine the structure at 21 Å resolution. This density map aided
construction of a homology-based model of sPvr and, in conjunction with the known
crystal structure of the virus, allowed determination of the binding site. The virion does
not change significantly in structure on binding sPvr in short incubations at 4°C. We infer
that the binding configuration visualized represents the initial interaction that is followed
by structural changes in the virion as infection proceeds. sPvr is segmented into three
well-defined Ig-like domains. The two domains closest to the virion (domains 1 and 2)
are aligned and rigidly connected, whereas domain 3 diverges at an angle of ~60°. Two
nodules of density on domain 2 are identified as glycosylation sites. Domain 1 penetrates
the "canyon" that surrounds the 5-fold protrusion on the capsid surface, and its binding
site involves all three major capsid proteins. The inferred pattern of virus-sPvr
interactions accounts for most mutations that affect the binding of Pvr to poliovirus.
We have also studied the thermodynamics of the native (N) virion to the altered
(A) particle transition. The altered particle results from the loss of viral protein 4 from the
capsid and other structural changes in the virion. The receptor was shown to behave like a
classic transition state theory catalyst for the N to A transition. The receptor accelerates
the rate of the transition by lowering the activation barrier 50 Kcal/mol. These results
confirm the prediction that the receptor acts much like an enzyme.
The accumulated data was used to define the basic rules of picornavirus receptor
interactions and to construct a model of genome release.
Table of Contents
List of Tables
iv
List of Figures
v
List of Abbreviations
vii
Acknowledgment
viii
Dedication
x
Chapter I. Introduction
1
Picornavirus crystal structures
2
Picornavirus solution structures
8
Poliovirus pathogenesis and tissue tropism
9
Poliovirus host range
11
The cellular poliovirus receptor
13
Early events in poliovirus infection
17
Kinetic and affinity analysis of Picornavirus-receptor interactions
21
Picornavirus-receptor structures
25
Viral capsid sequences that regulate receptor binding
28
Pvr sequences that contact virus
30
Hydrophobic antiviral agents
31
Chapter II. Materials and Methods
32
Cells and Viruses
33
Plasmid construction
33
Establishment of a stable cell line expressing sPvr
34
-i-
Protein expression, purification, and modification
35
Virus neutralization assay and determination of buffers for use with the optical
37
biosensor
Alteration assays
37
Binding of sPvr to poliovirus using an optical biosensor
38
Chapter III. Two Distinct Binding Affinities of Poliovirus for Its Cellular
39
Receptor
Introduction
40
Expression and purification of sPvr in mammalian cells
40
Virus neutralization and alteration activity of sPvr
41
Conditions and specificity of surface plasmon resonance
44
Kinetic and equilibrium affinity analysis
49
Chapter IV. Three-dimensional structure of poliovirus receptor bound to
59
poliovirus
Introduction
60
Visualization of poliovirus decorated with sPvr
60
The structure of the receptor fragment
61
Contact area on poliovirus
65
Model of the receptor fragment
66
Deglycosylated sPvr (dsPvr)
68
- ii -
Chapter VI. Discussion
78
Kinetics of poliovirus interaction with sPvr
79
Correlation of CryoEM structure with mutational data
83
An overview of the site of interaction
85
srr mutants possess varying degrees of resistance to sPvr
86
Recombinant sPvr and soluble Pvr purified from human serum neutralize
87
poliovirus at similar concentrations
Kinetic analysis of the effect of poliovirus receptor on viral uncoating: the
88
receptor as a catalysis
General rules for picornaviruses receptor interactions
93
Model of poliovirus interaction with cell surface
96
Conclusions and future directions
97
- iii -
List of Tables
1
Purification of sPvr
43
2
Kinetic and affinity parameters for sPvr binding to poliovirus type 1 at 20°C 54
3
Affinity for sPvr binding to poliovirus type 1 and abundance of each
56
binding class at different temperatures
4
srr mutants: level of resistance to sPvr, location of mutation, and phenotype
- iv -
91
List of Figures
1
Structure of poliovirus proteins
4
2
Schematic of poliovirus virion
6
3
Structures of the four Pvr proteins produced by alternative splicing
15
4
A model for poliovirus entry
22
5
A model of genomic RNA transfer across the cell membrane
23
6
Purity of recombinant sPvr expressed in mammalian cells
42
7
Neutralization of poliovirus infectivity by sPvr
45
8
Kinetics of sPvr-induced conformational changes of poliovirus
46
9
Example of raw sensorgram data
48
10
Effect of low pH treatment on poliovirus infectivity
50
11
Specificity of sPvr interaction with poliovirus on sensor chip
51
12
Corrected sensorgram overlays for the interaction of decreasing
53
concentrations of sPvr with immobilized poliovirus
13
Equilibrium binding sensorgrams and Scatchard analysis of the binding of
56
sPvr to immobilized poliovirus
14
CryoEM of poliovirus labeled with sPvr
62
15
Analysis of the Reconstructions
63
16
Trimming of an asparagine-linked precursor to a high-mannose structure
70
17
Purity of processed recombinant sdPvr expressed in mammalian cells
72
18
Neutralization of poliovirus infectivity by sdPvr and sPvr
73
19
CryoEM of poliovirus labeled with sdPvr
74
-v-
20
Neutralization of poliovirus infectivity by sCD155 and sPvr
75
21
Neutralization of wt and srr mutants
76
22
Locations of srr mutations in the capsid
89
23
Diagram of domain 1 of Pvr, with β-strand labeled, abutting inferred
90
contact segments of the capsid proteins
24
Arrhenius plot for the N to A transition in the presence and absence of sPvr
95
25
Bind, remodel, release model for poliovirus entry
100
- vi -
List of Abbreviations
bp
Base pair
BC
Known loops and strands in capsid proteins are
denoted by capitals
C’C’’
Putative loops and strands in Pvr or ICAM-1
domains are bold italic capitals
sdPvr
Deglycosylated sPvr
HRV
Human rhinovirus
ka
Association rate constant
kd
Dissociation rate constant
Kd
Dissociation constant
kD
Kilodalton
MPH
Murine poliovirus receptor homologue (gene)
Mph
Murine poliovirus receptor homologue (protein)
PVR
Poliovirus receptor (gene)
Pvr
Poliovirus receptor (protein)
sPvr
Soluble Pvr
sCD155
Soluble CD155 (sPvr isolated from humans)
- vii -
Acknowledgment
The definition of the word mentor, according to the Oxford English Dictionary, is
an experienced and trusted counselor. This word describes Vincent Racaniello well. He
guided me in a balanced manner and has been both scientifically critical and encouraging.
I wish to thank Saul Silverstein, Jeremy Luban, Stephen Goff, and Hamish Young for
their encouragement and helpful advice over the years. Additionally, I would like to
thank Saul Silverstein for his avuncular spirit, which added color to the everyday.
I would like to thank James Hogle, Alasdair Steven and members of their
laboratories at Harvard University and The National Institutes of Health who have been
valuable collaborators. In particular, David Belnap and Simon Tsang have been generous
with their insights, protocols and unpublished data.
I am grateful to Roselyn Eisenberg, and Gary Cohen for allowing me to work in
their laboratory and use their Biacore biosensor. In particular, I am appreciative of Ann
Rux, from the Cohen lab, for assisting with Biacore data analysis. I am thankful to
Shazad Majeed and Peter Kwong, from Colombia University, for collaborating with me
to make sdPvr and for many interesting conversations.
I am grateful to my colleagues in Vince’s lab both past and present. Michael
Bouchard, Yangzang Dong, Alan Dove, Ornella Flore, Julie Harris, Scott Hughes, Steven
Kauder, Du Lam, Sa Liao, Yi Lin, Carl Pavel, Amy Rosenfeld, Juan Salas-Benito,
Melissa Stewart, and Suhua Zhang have contributed to my scientific and personal
development through thoughtful conversations, advice, humor, and friendship. Members
of the Silverstein, Young and Efstratiadis labs have been generous intellectual
- viii -
companions and they have my sincere thanks. From these labs, I especially thank
Christopher Newhouse and Iaonnis Dragatsis for their friendship.
Finally, I would like to thank my family and friends, especially my brother,
Jonathan, and my wife, Yvonne, who have been patient, encouraging, and loving from
the beginning of this endeavor.
- ix -
Dedication
In memory of Lucille, who experienced the beginning of this work, understood its
difficulty, smiled, and named sPvr “The Great White Protein”
-x-
Chapter I. Introduction
-1-
Picornavirus crystal structures
Picornaviruses are naked icosahedral viruses that are typically composed of sixty
copies of four polypeptide chains, designated: VP1 (viral protein 1), VP2, VP3, and VP4.
VP4 is an internal capsid protein that does not have access to the surface of the protein
shell in the crystal structure. VP1, VP2, and VP3 form the exposed surface of the protein
shell; these three capsid proteins contain a common central structural element known as
β-barrel jelly roll (Figure1). VP1-3 differ in the loops that contact the β-strands and in the
amino and carboxy terminal extensions. The surface peaks are formed by loops
connecting the β-strand and the carboxy terminal extensions that are exposed to the
exterior of the virion. A byzantine network formed by the amino terminal extensions of
VP1, VP3, and VP4 characterizes the interior of the capsid. The β-barrel jelly rolls of
picornavirus structural proteins are similar in structure to the core domain of capsid
proteins of a number of plants, insect, and vertebrate (+) strand RNA viruses, such as
tomato bushy stunt virus (TBSV).
The crystal structure of poliovirus has revealed that the proteinacious coat is
organized in a hierarchical manner of repeating units. The simplest is the promoter, a
heteromeric structural unit of the poliovirus capsid that contains one copy of each of the
peptide chains (63). Five copies of VP1 surround the five-fold axis of symmetry, three
copies of VP2 and VP3 alternate around the three-fold axis, and VP4 is located on the
interior of the virion. The capsid surface is characterized by three prominent features: a
mesa, the highest peak located at the five-fold axes of symmetry; the propeller, a smaller
peak at the three-fold axes of symmetry; and the canyon, a surface depression that
-2-
arrangements exist in many other picornaviruses, such as rhinovirus (122). Conservation
of capsid structure among picornaviruses suggests information about one virus may be
relevant to another. Furthermore, the features and mechanisms of virus entry, such as,
receptor binding loci, receptor binding behavior, and the uncoating process may also be
conserved throughout the picornavirus family.
At the interface between five-fold related protomers are structural features that
regulate conformational transitions during virion assembly, as well as, cell entry. The
temperature-sensitive (ts) phenotype of the Sabin vaccine strain of poliovirus type 3
(P3/Sabin) is caused by a Phe to Ser mutation at residue 91 of VP3. In the virion, residue
91 of VP3 is on the surface in the canyon near the interface between five-fold related
protomers (103). Results of temperature shift experiments demonstrated that the
temperature sensitivity is expressed during assembly, specifically at the protomer-topentamer step (103); these results suggest that the mutations at non-permissive
temperatures appear to affect the correct folding and stability of the capsid proteins and
early assembly intermediates. Revertants of this temperature sensitive mutation have
allowed for identification of capsid structures involved in assembly and cell entry. These
structures are the hydrocarbon-binding pocket of VP1, the β-tube, and the seven-stranded
β-sheet.
An enigmatic feature of picornavirus capsid structure is a hydrocarbon-binding
pocket in VP1. It is located beneath the canyon floor, near the interface between
protomers and is believed to modulate receptor-mediated conformational transitions
during entry. The pocket is normally occupied by a sausage-like hydrocarbon molecule
-3-
Figure 1:
Structures of poliovirus proteins
A representation of the physical features of the polypeptide chain in a β-barrel
jelly roll is shown at the top left. The β-strands, indicated by arrows, form two
antiparallel sheets juxtaposed in a wedgelike structure. The two α-helices (purple
cylinders) that surround the open end of the wedge are also conserved in location and
orientation in these proteins. The VP1, VP2, and VP3 proteins each contain a central βbarrel jelly roll domain. However, the loops that connect the β-strands in this domain of
the three proteins vary considerably in length and conformation, particularly at the top of
the β-barrel domain, which, as represented here, corresponds to the outer surface of the
capsid. The N- and C-terminal segments of the proteins, which extend from their β-barrel
cores, also vary in length and structure. The very long N-terminal extension of VP3 has
been truncated in this representation (Adapted from (126)).
-4-
-5-
5x
3x
2x
Figure 2: Schematic of poliovirus virion, showing the names and locations of capsid
proteins and genomic RNA (From (126)).
-6-
that resembles sphingosine, but it remains to be verified chemically. Evidence that the
hydrocarbon-binding pocket is involved in conformational transitions during virion
assembly and cell entry comes from studies of poliovirus mutants. Non-temperature
sensitive (ts) suppressor revertants of P3/Sabin have been isolated and characterized (92).
The suppressor mutation at a second site introduced a leucine in place of the
phenylalanine at VP1-132. The side chain of residue VP1-132 is in a pocket that binds
the endogenous lipid ligand in the hydrophobic center of VP1. Suppressing mutations are
located in, or near, structures believed to regulate virion stability and conformational
transitions, the interface between five-fold related protomers and the hydrocarbonbinding pocket (44). Suppressing mutations in the pocket may stabilize the interface
during pentamer assembly at the non-permissive temperature by enhancing hydrocarbon
binding.
Two additional structures believed to be important for virion assembly are a ßtube and a seven-stranded ß-sheet. The ß-tube is a five-stranded tube of parallel ß
structure that is formed by the interaction of the amino termini of five copies of VP3 on
the inner surface of the capsid at the five-fold axis of symmetry (63). The ß-tube is
flanked by five copies of a three-stranded ß-sheet formed from the amino termini of VP1
and VP4 (63). The amino terminus of VP4 is myristoylated and is thought to direct the
interaction between VP3 and VP4 (32). This extensive network between five-fold related
protomers can only form upon pentamer assembly and is believed to stabilize pentamers.
The seven-stranded ß-sheet is formed near the three-fold axis by four ß-strands of VP3
and the amino terminal extensions of VP1 from one pentamer and the amino terminal
-7-
relies on the association of pentamers into larger structures and presumably stabilizes the
pentamer-pentamer interaction. A third class of the aforementioned P3/Sabin ts
suppressing mutations is located in the seven-stranded ß-sheet (44) and may counteract
the effect of the original ts mutation by stabilizing assembly intermediates.
Picornavirus solution structures
Analyses of solution structures of poliovirus and rhinovirus have demonstrated
dynamic capsid configurations. This is not surprising because a dynamic capsid is
necessary for many aspects of the viral life cycle. Early entry related events such as cell
attachment, cell entry, and nucleic acid release require many structural transitions of the
viral surface. Rhinovirus has been shown to have a dynamic capsid structure; time course
mass mapping has revealed that capsid proteins, which are internal in the crystal
structure, are transiently exposed in solution at 25 °C (86). In this comparative study,
treatment with WIN 52084, an antiviral agent that inhibits rhinovirus entry, resulted not
in local capsid conformational changes in the drug-binding pocket, but a global
stabilization of the entire viral capsid (86). Similarly, the poliovirus capsid is a dynamic
assemblage that is capable of undergoing conformational transitions at 37 °C.
Immunoprecipitation analysis demonstrated that it is possible to neutralize poliovirus
with antibody directed against internal capsid residues. Neutralization is a result of
reversible exposure of these normally internal sequences at 37 °C and antibody binding
(87). Since the sequences reversibly exposed at 37°C in the 160S particle are the same as
-8-
conformational dynamics may play a role in cell entry (87).
Poliovirus pathogenesis and tissue tropism
The mode of poliovirus transmission is by the fecal-oral route (21). Virus initially
multiplies in the lymphoid tissue of the pharynx and gut, which results in a transient
viremia that facilitates spread of the virus to other susceptible tissues, such as brown fat
and muscle (21). In most natural infections viral replication in the gut leads only to a
transient viremia. Replication in secondary sites is thought to be essential for the
establishment of a persistent viremia, which is necessary for spread of the virus to the
central nervous system (CNS). In approximately 1% of infections the virus enters and
replicates in the central nervous system, causing lesions in motor neurons of the spinal
cord, the brainstem, and the motor cortex (127). The characteristic flaccid paralysis of
acute paralytic poliomyelitis results from lysis of motor neurons in the spinal cord.
Within an infected host, viral replication is limited to certain tissues and cells.
The conventional view was that poliovirus tissue tropism was determined solely by
receptor expression patterning or accessibility of cells to the virus (64). However, there
is evidence that tissue tropism is determined by cellular factors other than the poliovirus
receptor (Pvr), either at the cell surface or in the cytoplasm (45, 102, 119, 137). The
development of a transgenic mouse model has advanced the understanding of poliovirus
pathogenesis and tissue tropism (76, 120). However, unlike the case for poliovirus
infection in humans, poliovirus replication is not detected in the alimentary tract of
transgenic mice after oral inoculation. To determine whether Pvr is the sole determinant
-9-
levels of Pvr in M cells and enterocytes were generated (152). Poliovirus was unable to
replicate in the small intestine of this transgenic mouse line. These results indicate that
Pvr expression is not the sole determinant of the resistance of the mouse intestine to
poliovirus replication. Other cellular factors may influence the ability of poliovirus to
enter or replicate in cells of the mouse alimentary tract (152).
The internal ribosome entry segment (IRES), a cis-acting genomic element at the
5’ end of the RNA genome, may play a role in determining tissue tropism of the virus.
Cellular proteins bind to IRES sequences, some of which have been shown to be essential
for IRES activity in vitro (14, 20, 59, 70, 101). Several lines of evidence support the
hypothesis that the IRES participates in the tropism of the virus. A main determinant
involved in neurovirulance attenuation of the Sabin vaccine strain of poliovirus type 3
was mapped to the IRES (1). Translation from the IRES of attenuated strains in vitro
were shown to be specifically inhibited in cell lines of neuronal origin (55). Furthermore,
poliovirus neuropathology in a mouse model was eliminated when the IRES of this virus
was replaced by the IRES of rhinovirus (53). Lastly, translation from the IRES of
hepatitis A virus in vitro was found to be stimulated 12-fold when fresh liver extracts
were added to the assay mixture (50). In contrast to these findings, the IRES of the
Theiler’s virus does not determine its tropism; IRES activity, in the context of a
bicistronic construct, was detected at similar levels in vivo in all examined tissues of the
mouse (135).
- 10 -
Humans are the only known natural hosts of poliovirus. Monkeys are also highly
susceptible to poliovirus when they are inoculated with virus directly into the central
nervous system . Other animal species are generally not susceptible to poliovirus. This
characteristic species specificity of poliovirus has meant that monkeys have been used as
the only animal model for the study of poliovirus neurovirulance and safety testing of
oral poliovirus vaccines.
The majority of poliovirus strains are host-restricted; for example, P1/Mahoney
causes paralysis in primates, but not in mice. In contrast, some poliovirus strains naturally
cause paralysis in mice and others have been adapted; for example, the Lansing strain of
poliovirus type 2 (P2/Lansing) was adapted to cause paralysis in mice after serial passage
in the CNS of cotton rats (5). Intracerebral or intraspinal inoculation of mice with
P2/Lansing causes a disease that resembles human poliomyelitis clinically and
histopathologically (69, 83), but orally inoculated virus does not replicate in mice (83), or
in mouse L cells (106).
Transgenic mice expressing the human poliovirus receptor are susceptible to
infection by host-restricted poliovirus strains (120), demonstrating that host restriction is,
in part, at the level of the virus-receptor interaction. Molecular analysis of viral host
range determinants has provided information on the virus-receptor interaction. Intertypic
recombinants between mouse-adapted P2/Lansing and host-restricted P1/Mahoney
demonstrated that the ability of Lansing virus to cause paralysis in mice is due to the viral
capsid (83). A chimeric virus was generated to map the region of the capsid that confers
mouse virulence. The region was narrowed to an eight amino stretch of VP1, the B-C
- 11 -
conferred mouse-virulence to P1/Mahoney (96, 109). Therefore, the VP1 B-C loop is an
important determinant of the mouse virulence of P2/Lansing and the host-restriction of
P1/Mahoney.
The mechanism by which the B-C loop modulates host range is unclear. One
explanation for the importance of this highly exposed portion of the viral capsid in
regulating mouse-adaptation is that mouse-virulence is determined by the ability of the
virus to attach to a specific receptor in the mouse nervous system; more specifically, host
range may be determined by receptor recognition, and the B-C loop might be the binding
site for a mouse receptor. The prominent exposure of the P2/Lansing B-C loop at the
five-fold axis on the virion surface (63, 85), and the finding that few changes in the B-C
loop are compatible with mouse neurovirulence (83, 108) are consistent with this
hypothesis. However, host range determinants that suppress B-C loop mutations and
confer mouse neurovirulence to P1/Mahoney have been identified on the interior of the
capsid in the amino terminal network (36, 108), and it is unclear how they might directly
regulate receptor recognition.
Another possibility is that mouse neurovirulence is not determined by receptor
binding. It is possible that there is a relationship between mouse adaptation of poliovirus
and the involvement of VP1 B-C loop conformational changes required for infectivity
(150). The mouse receptor may be able to bind to all three serotypes of poliovirus, but it
may not be capable of inducing the structural changes in P1/M and P3/S required for
infectivity. It is also conceivable that theVP1 B-C loop of P2/L allows the virus to enter
by a different route of infection. The VP1 B-C loop conformation may have a dramatic
- 12 -
loops and possibly the flexibility of the capsid (44, 150). Structural ‘cross talk’ may exist
between VP1 B-C loop and conformations of other nearby sites. This situation would be
similar to one observed for the foot-and-mouth disease virus. It has been proposed that
the flexibility of the antigenic/receptor-binding loop (G-H loop of VP1) of foot-andmouth disease virus not only directly affects antigenicity, but also influences host cell
interactions-possibly by structural ‘cross talk’.
The cellular poliovirus receptor
Recognition of a cell surface receptor is the first step in infection of cells by
animal viruses. The poliovirus receptor is a member of the immunoglobulin superfamily
that is used by all three viral serotypes to initiate infection of cells (102). The receptor is a
type I integral membrane protein with a primary amino acid sequence that encodes an Nterminal secretion signal, three extracellular immunoglobulin (Ig)-like domains, a
transmembrane region, and a cytoplasmic domain. There are four putative alternately
spliced mRNA variants of the receptor hnRNA: two variants lack the transmembrane
region and appear to be secreted; and, two variants that serve as poliovirus receptors
encoding polypeptides of 392 and 417 A.A., differing in the lengths of their cytoplasmic
domain. (Figure 3) (75, 102). The ectodomain of each of the forms contains 8 putative Nlinked glycosylation sites (102) that shift the molecular mass from the predicted 43 or 45
kDa, for the membrane bound forms of Pvr, to a predominant species of about 80 kDa on
a SDS-PAGE gel. Post-translational modification of the receptor is not necessary for
entry of the virus, although destruction of the first glycosylation site by mutagenesis
- 13 -
glycosylation may partially occlude virus docking (154).
The prediction that the extracellular portion of Pvr contains three Ig-like domains
is based on consensus homology. An Ig-like domain is defined by sets of conserved
residues that permit the polypeptide to from a globular tertiary structure called the
antibody-fold. Antibody folds contain 70-110 amino acids that form two sets of β-pleated
sheets, each consisting of three or four anti-parallel β strands of five to ten amino acids in
length (147). The interior of the fold is lined with hydrophobic residues, which alternate
with outer facing hydrophilic residues. Intra-chain disulfide bonds are usually present on
every domain and contribute to the stability of the structure (147). Antibody folds are
broadly classified as V- or C-like, based on their relative homology to either the variable
or the constant domains of immunoglobulin molecules. Domain 1 of Pvr is V-like while
the other two more membrane proximal domains are C-like (102). V-like domains are
generally larger than C-like domains, and usually contain an extra pair of β strands.
A number of Pvr-related proteins have been identified, but not all of them
function as receptors for poliovirus. As a group, most of these proteins serve as receptors
for a number of viruses. All Pvr-related proteins are members of the immunoglobulin
superfamily. Agm1 and Agm 2 receptors, isolated from African green monkeys, are
functional receptors for poliovirus (78). Pvr mediates the entry of pseudorabies virus
(PRV) and bovine herpesvirus-1 (BHV-1), but has no activity for herpes simplex virus
(HSV) strains. Two other Pvr-related proteins, which do not serve as receptors for
poliovirus, were originally designated poliovirus receptor-related protein 1(Prr1) (91) and
poliovirus receptor-related protein 2 (Prr2) (43), serve as HSV entry mediators. Prr2 was
- 14 -
H 20 A
45 kDa
H 20 B
43 kDa
H 20 A∆1
40 kDa
H 20 A∆2
39 kDa
Figure 3:
Structures of the four Pvr proteins produced by alternative splicing.
The membrane bound forms of Pvr are labeled H 20 A and H 20 B. The secreted forms of
Pvr are labeled H 20 A∆1 and H 20A∆2. The putative Ig-like domains are represented by
circles; leftmost and largest is the V-like domain. The green boxes represent
transmembrane regions. The pink line denotes the C terminal region of the H 20 B
variant, which is unique.
- 15 -
mediator (HVEM) for entry. It has been renamed herpes virus entry protein B (HveB)
(144). An alternate name for HveB is Mph, mouse poliovirus receptor homologue. Prr1
was found to serve as an entry receptor for HSV-1, HSV-2, PRV, and BHV-1 and was
recently designated herpes virus entry protein C (HveC) (48).
Pvr’s cellular function and its role in development are unknown. The cytoplasmic
domain of one of the Pvr isoforms is phosphorylated at a serine residue, possible by
calcium/calmodulin kinase II (18). Insights into the function of Pvr can be gained by
studying the function of a Pvr homologue in mice and in in vitro culture systems. Mph is
a Ca2+-independent homophilic cell adhesion molecule. The cytoplasmic tail of Mph
interacts with 1-afadin, an actin filament (F-actin)-binding protein with one PDZ domain.
This interaction creates a link between the cytoplasmic tail of Mph to the actin
cytoskeleton. The interaction of Mph with 1-afadin is not essential for its cis-dimerization
or trans interaction (104), but is essential for the colocalization of MPH and 1-afadin with
E-cadherin at cell-cell adherence junctions (141). Recently, to provide information on the
function of Pvr family members, Mph was targeted for disruption in the mouse.
Disruption of both alleles of the Mph gene resulted in morphologically aberrant
spermatozoa. The spermatozoa contained defects in nuclear and cytoskeletal morphology,
mitochondrial localization, and F-actin distribution (22). These results suggest signaling
through Mph may be crucial for the cytoskeletal organization and reorganization that
occur during spermatogenesis (22).
- 16 -
A model of poliovirus entry is displayed in figure 4. Shortly after poliovirus binds
to cell surface Pvr, it releases its genomic contents into the host cell cytoplasm. Pvr plays
two roles in virus entry: first, the receptor acts as a tether to which the viruses attach and
concentrates particles on the cell surface; second, it facilitates the viral uncoating step by
inducing dramatic structural changes in the virus particle (51). When poliovirus is bound
to cells at 37oC, the major population of virus is eluted as a conformationally altered form
known as the ‘A’ particle. A minor population also elutes off the cell surface and is
termed either the 80S particle or the empty capsid.
The altered form of the virion is also referred to as the 135S particle, because it
sediments at 135S on a sucrose gradient, compared to 160S for the native virion. This
conformational alteration results in the release of the internal capsid protein VP4 and the
externalization of the N terminal extension of VP1 (46). The N-terminus of VP1 allows
the altered particles to associate with lipid bilayers (46), it is also thought to be involved
in formation of a pore. Multiple N termini, which have amphipathic helical character,
insert into the cell membrane producing a pore through which the viral genome may pass
to enter the host cell cytoplasm (Figure 5).
The A particle has been proposed to be an essential intermediate in the entry of
poliovirus into cells (46). Isolation of cold adapted mutants (Ca) provide evidence against
the A particle as an intermediate in the entry process. The mutants infect efficiently at 25°
C without formation of 135S particles. These mutants can form 135S particles at 37°C
(42). These data suggest formation of 135S particles is not required for poliovirus
replication.
- 17 -
high concentrations of the 135S particle are capable of infecting cells in a receptor
independent fashion (38). Moreover, binding of 135S-antibody complexes to the Fc
receptor attached to the cell surface increases the infectivity of these particles by 2 to 3
orders of magnitude (66). Thus, the low efficiency of infection by 135S particles is due,
in part, to the low binding affinity of these particles. Furthermore, it has been shown that
there is an additional stage in the entry process that is associated with RNA release. This
Pvr independent stage occurs after formation of the 135S particle and is rate limiting
during infection at 37°C, but not at 26°C. This study also demonstrated that during
infection at 26°C, the rate-limiting step is the Pvr-mediated conversion of wild-type 160S
particles to 135S particles. From these data, it has been suggested that infection at 26° C
by the cold-adapted viruses allows the 135S particles to be formed, but they fail to
accumulate to detectable levels because the subsequent post-135S particle events occur at
a much faster rate than the initial conversion of 160S to 135S. This indicates a model in
which the 135S particle as an intermediate during poliovirus entry is possible (66).
In the model presented in figure 4, after the virus forms the A particle some
particles may release their RNA. The coincident release of the RNA genome and VP4
from the particle results in the formation of the 80S particle (Figure 4). This process is
known as uncoating.
Tomato bushy stunt virus (TBSV) has a capsid structure similar to poliovirus and
undergoes a well-characterized particle expansion that may be similar to poliovirus
alteration. Exposure of TBSV to an alkaline environment devoid of cations causes
reversible expansion of the virus (81). Viral enlargement is controlled by an interface
- 18 -
intermediates similar to the intermediates formed by poliovirus. Resolution of the TBSV
crystal structure, in conjunction with biochemical studies of the expanded particles, has
revealed that the buried amino termini of capsid subunits are extruded through holes in
the disrupted interfaces (Harrison et al., 1986). Based on these observations, analogies
between TBSV expansion and poliovirus alteration have been made.
It has been suggested that poliovirus is similar to the TBSV and the analogous
interface between five-fold-related protomers controls poliovirus alteration. Interestingly,
capsid protein VP4 and the amino terminus of VP1 are located immediately below the
interface (63) and appear to be hydrogen-bonded to each other, providing a mechanism
for the coordinated release of these internal components during alteration (44). A
poliovirus VP4 mutant has been isolated that binds Pvr and undergoes alteration, but is
blocked at a subsequent entry step (107). Other mutants with deletions in the amino
terminus of VP1 have delayed RNA release (74). These mutants lend support to the idea
that VP4 and the amino terminus of VP1 are involved in early entry events. Interestingly,
there are significant differences between the 135S structure and the expanded structure of
the TBSV. The expanded forms of the plant viruses have holes large enough to be
visualized by low resolution; however, the gaps between VP1 subunits are barely large
enough to allow passage of an extended polypeptide chain. This suggests that during the
poliovirus alteration process the holes are larger (12).
The subcellular location where the virus releases its RNA into the cytosol is
unclear. Although significant progress has been made, the confounding problem in
determining the subcellular location of entry is partly due to the inefficiency of virus
- 19 -
determine the subcellular location of viral genome release by standard viral metabolic
labeling and cell fractionation techniques. Electron micrographs of poliovirus particles
have been observed in coated pits and vesicles after adsorption, suggesting that virus
might enter via receptor-mediated endocytosis (151). Further studies have been
preformed to determine if poliovirus entry is dependent on clathrin, the transmembrame
or cytoplasmic regions of Pvr, or a low-pH step to enter the cytoplasm.
Virus entry studies were performed using HeLa cell lines that express a mutant
form of dynamin; these cell lines specifically block the formation of clathrin-coated pits
and vesicles without other pleotropic effects (6). It was demonstrated that human
rhinovirus 14 depends on the clathrin pathway to enter Hela cells, whereas poliovirus
does not (40). Moreover, poliovirus is able to infect cells using recombinant Pvr receptors
that have different primary amino acid sequences in the transmembrane and cytoplasmic
regions substituted from other cell surface molecules. These results indicate that Pvr
amino acid sequences in the cytoplasmic and transmembrane regions are not necessary
for infection in cultured cells (106, 131, 132).
The requirement of a low-pH step during poliovirus entry has been investigated
by using the macrolide antibiotic bafilomycin A1, which is a powerful and selective
inhibitor of the vacuolar proton-ATPases; it was demonstrated that poliovirus infection is
not affected by the antibiotic (112). The presence of lysosomotropic agents such as
chloroquine, amantadine, danaylcadaverine, and monensin during poliovirus entry did not
inhibit infection (112). These data support the hypothesis that poliovirus does not depend
on a low-pH step to enter the cytoplasm (112).
- 20 -
135S and 80S particles have been determined to 22 Å resolution (12). Based on these
structures, the tectonic plate model has been formulated to describe the structural changes
the virus undergoes during conversion catalyzed by the receptor. Domain movements of
up to 9Å were detected that create gaps between adjacent subunits. The gaps formed
where VP1, VP2, and VP3 subunits meet and are predicted to be the site of emergence of
VP4 and the N terminus of VP1. In the transition, VP4 and the N termini of VP1 are
extruded from the bottom of the canyon and arranged around the outside of the mesa,
where five copies of the predicted amphipathic helices of the N terminus of VP1 would
be ideally positioned for membrane insertion (12). It is possible that the N-terminal
myristate of VP4 and perhaps other regions of VP4 also may be imbedded into the
membrane. This could facilitate insertion of the VP1 N termini into the membrane (12).
To create a channel at the five-fold axis, a transmembrane pore is formed and the VP3
plug is moved out of the way (Figure 5). The genome then exits the particle and enters
the cytoplasm. During this process, the 80S particle is formed by shifts of the VP1, VP2,
and VP3 subunits and the fivefold plug is restored to its original position (12).
Kinetic and affinity analysis of picornavirus-receptor interactions
Kinetic and affinity parameters of the various viruses with soluble forms of their
receptors have been determined. Such parameters are important because they describe the
interaction of virus with receptor, which enables a better understanding of the reaction
and its comparison to other systems. The results of such studies, together with structural
- 21 -
Poliovirus Entry
native
virion
160S
extracellular
A particle
o
>33 C
Pvr
A particle
uncoating
empty capsid
80 S
Figure 4. A model for poliovirus entry. 160S native virions bind to Pvr on the cell
surface and, at temperatures greater than 32°C, undergo the receptor-mediated
conformational transition to 135S altered particles (A particles). While a large
percentage of A particles elute from the cell, some remain cell-associated and release
their RNA into the host cell cytoplasm. The cellular location of uncoating, which yields
the 80S particle, is unclear.
- 22 -
Figure 5: A model of genomic RNA transfer across the cell membrane. VP1, VP2,
VP3, and VP4 are colored cyan, yellow, red, and green, respectively. In this model, the
beta-tube of VP3 (red) forms a plug at the fivefold axis that separates the virus interior
from the outer surface. Attachment of the 160S particle (left) to the poliovirus receptor
(three gray circles) triggers conversion to the 135S form (center). As conversion
commences, cell attachment is mediated by externalized VP4 (green tubes) and the N
termini of VP1 (blue tubes). The N termini emerge from the bottom of the canyon and
extend along the sides of the fivefold mesa towards the apex. After the N-terminal helices
of VP1 have inserted into the membrane, they rearrange to form a pore (right). To permit
the RNA (purple tube) to pass through the pore into the cytoplasm, the VP3 beta-tube
(red rectangle) may shift on its 40-residue tether (red tube) and the VP1 barrels could
splay farther apart (From (12).
- 23 -
events in infection (61, 80, 125, 153).
Surface plasmon resonance (SPR) has been used to study affinity and kinetics of
the interaction of echovirus 11 with its cellular receptor decay-accelerating factor
(CD55). This virus-receptor interaction is monophasic: there is a single affinity (Kd) for
the interaction. The interaction was reported to be similar to cell-cell recognition
molecules with a relatively low affinity of Kd=3.0 µM as a result of a very fast
dissociation rate (ka ~105 M-1.s-1 kd~0.3 s-1) (84). In contrast to the Echovirus 11 virussoluble receptor interaction, rhinovirus-sICAM interaction in solution and using surface
plasmon resonance were determined to be biphasic: i.e., there are 2 affinities of the virus
for the receptor. These SPR studies have shown that there are two different association
rates and a single dissociation rate for the interaction. Each class of binding site
comprises 50 % of the total at 20°C, with association rate constants of 2450 and 134 M-1s1
. The off rate for human rhinovirus 3 was 1.7 x 10-3s-1 to yield a calculated dissociation
constant of 0.7 and 12.5 µM (28). It has been proposed that the 2 binding classes may be
a consequence of virus breathing and the receptor binding site may exist in at least 2
different conformational states. The single dissociation and biphasic association rates
indicate a unique interaction site with variable conformation or accessibility to the
receptor (26). Interestingly, poliovirus demonstrates two modes of binding to the surface
of cells that express cell surface Pvr, a lower and a higher affinity class (8). Each of the
classes could represent two different receptors, different states of the same receptor, or
two states of the virus.
- 24 -
The "canyon hypothesis" has been proposed to explain binding of rhinovirus to its
cell receptor, intercellular adhesion molecule-1 (ICAM-1) (121). The structural
similarity between poliovirus and rhinovirus raises the possibility that the poliovirus
canyon may be the binding site for Pvr. The first draft of the canyon hypothesis
predicted that conserved residues on the floor of the HRV-14 canyon bind to the cell
receptor, but the small dimensions of the canyon should limit accessibility to antibodies.
Furthermore, HRV-14 morphology should allow residues at the top of the canyon to
mutate freely without affecting virus receptor interactions (121). However, the latter
aspect of the hypothesis was nullified by the determination of the three-dimensional
structure of intact human rhinovirus 14 complexed with Fab fragments, Fab17-IA.
Fab17-IA penetrates deep within the canyon and overlaps the region the receptor binds.
Hence it is unlikely that viral canyon quaternary structure evolved merely to evade
immune recognition. Instead, the shape and position of the receptor binding region on
rhinovirus probably dictates receptor binding and subsequent uncoating events. (139).
Availability of the three-dimensional crystal structure of both rhinovirus and the
first two domains of ICAM-1 has made it possible to investigate their interaction (10, 11,
79, 122). Similar to poliovirus genetic data (33, 35, 88), rhinovirus studies support the
hypothesis that the canyon contains the binding site for ICAM-1. Unfortunately, no
atomic resolution crystal structure of the complex between rhinovirus and its receptor is
available. However, low-resolution cryoEM image reconstructions of complexes of
soluble ICAM-1 fragments with HRV16 (111), HRV14 (111) and HRV3 (149) have been
determined. Complexes of the first two domains of ICAM-1 with HRV16 and HRV14
- 25 -
ICAM-1 fragment has a dumbbell-like shape in these reconstructions (111) and is
oriented roughly perpendicular to the viral surface. Atomic coordinates from the ICAM-1
receptor fragment (9) and HRV16 (110) crystal structures have been used to fit a
molecular model into the CryoEM image reconstruction (10). The modeled interaction
that shows the variable loops of the receptors BC, DE, and FG penetrate deep into the
canyon, and the short CD loop of ICAM-1 lies against VP2 of HRV16, on the “south”
rim of the canyon. The footprint of ICAM-1 on the HRV16 surface is essentially the
same as HRV 14, and the analysis of the charge distribution on the two interacting
surfaces shows remarkable complementarity (79), indicating that surface charge may play
an important role in rhinovirus–ICAM-1 recognition.
Coxsackievirus A21 (CAV21), like HRVs, is a causative agent of the common
cold. It uses the same cellular receptor, ICAM-1, as does the major group of HRV (133).
It can also bind to decay-accelerating factor, although without causing infection (134).
Interestingly, CAV21 differ from HRVs in that it is stable at acidic pH. Recently, the
cryoEM image reconstruction has been reported, at 26 Å, of CAV21 complexed with
ICAM-1(148). The rendering demonstrates that domain 1 of ICAM-1 binds to a similar
site as major group HRVs in the CAV21 canyon vicinity.
Foot-and-mouth disease virus (FMDV) causes a highly infectious disease in
cloven-hooved animals, which can be economically devastating. FMDV enters cells
through receptor-mediated endocytosis followed by binding acid pH-dependent release
and translocation of RNA across the endosomal membrane(24, 37, 98). The predominant
cell surface ligand is heparin sulfate (HS) for certain strains of O1 FMDV; its attachment
- 26 -
for O1 FMDV bound to HS has revealed the binding site is a shallow depression, the pit,
on the virion surface, located at the junction of the three major capsid proteins, VP1,
VP2, and VP3 (47).
Human rhinovirus 2 is a minor group rhinovirus that binds to the very low-density
lipoprotein (VLDL)-receptor (VLDL-R), a member of the LDL-receptor family. A cryo
EM and three-dimensional image reconstruction of the HRV2 and soluble fragments of
the VLDL-R complex have been determined to 15Å. Surprisingly, the receptor fragments
bind sites on the virus very different from major group rhinoviruses. The minor group
receptor binds to the star-shaped mesa on the 5-fold axis rather than in the canyon (60).
The difference in binding loci may begin to explain differences in uncoating
mechanism between major and minor group rhinoviruses. More specifically, binding of
ICAM-1 to HRV14 initiates rapid uncoating at physiological temperature and at neutral
pH without the need for any cellular components (65, 97). In contrast, binding-ldl
receptors to HRV2 does not directly promote uncoating (54, 93-95). Internalization of
HRV2 into acidic an endosomal compartment is required for uncoating and transfer of
viral RNA into the cytosol (115, 116, 130). It has been suggested that ICAM-1 binds to
the major group HRV in a two-step mechanism (26, 79), in which the Cryo EM
reconstructions of HRV complexed with soluble ICAM-1 fragments represent the initial
recognition event. In a subsequent step, the receptor could move slightly to allow the
north wall of the canyon, consisting of VP1 residues, to bind to domain D1. The
resulting conformational change in the virion may move VP1 away from the 5-fold axis,
thereby opening a channel at the pentamer vertex through which the N termini of VP1,
- 27 -
occur for HRV2 because its receptor binds to the peak at the five-fold axis.
Viral capsid sequences that regulate receptor binding
A detergent-solubilized form of the poliovirus receptor has been produced from
insect cells, expressing a membrane bound form of the receptor. These solubilized forms
of the receptor bind the virus and are capable of blocking infectivity and inducing the
conversion of the 160S to the 135S and 80S products (71, 154). This system was used to
identify poliovirus capsid residues involved in the virus-receptor interaction by isolating
spontaneously occurring viral mutants resistant to neutralization by solubilized receptor,
but remain infectious in cultured cells (33, 71). Neutralization by detergent-solubilized
receptor, or membrane bound receptor, is dependent on two processes that are
interrelated: receptor binding and conversion.
21 srr mutants were isolated each containing a single mutation located on the
surface or the interior of the capsid (Table 4, Figure 22). One of the mutants contained a
double mutation that displayed a very different phenotype. The single mutants loosely fit
into one of two categories (31, 33). One class of mutants have significantly reduced
affinity for the receptor on the surface of cells with or without reduced ability to undergo
alteration after binding to cells. A second class of mutants has moderately reduced
affinity for the receptor on the cell surface in addition to, a reduced ability to undergo
alteration after binding to cells. The single double mutant has a phenotype that is not
easily explained; it binds receptor with a similar affinity as wild-type virus and has an
increased ability to undergo alteration upon interaction with the surface of susceptible
- 28 -
or the interface between fivefold-related promoters (Table 4). The locations of the
residues altered in these mutants strongly implicate the canyon in receptor contact (Figure
22). Changes in residues 1226, 1228, 1231 and 1234, which are located at the surface of
the virus in the canyon, are mutations that reduce binding affinity to the cell surface.
These mutations may reduce the binding affinity by interfering with receptor contact.
Mutations at internal capsid residues also reduce binding affinity. These residues are not
likely to contact the receptor directly, but may affect the binding in two ways: (1) they
may distort the primary site of interaction by steric interference; or, (2) they may impede
the poliovirus or the receptor from undergoing the necessary structural rearrangements
necessary to reach a final bound state.
Further information on capsid residues that regulate interaction comes from the
analysis of adapted viral mutants that grow on cells expressing mutant forms of Pvr that
do not bind wild-type P1/Mahoney. Pvr mutants were constructed by substituting
residues of Pvr with corresponding residues from Mph. Since Mph is not a poliovirus
receptor, the described receptor mutagenesis locates regions of Pvr important for
poliovirus binding. Three cell lines were derived that cannot bind wild-type virus;
however, viral variants were readily isolated that can utilize the mutant Pvrs to infect
cells (105). These viral mutants have expanded receptor recognition because they are still
able to utilize wild-type Pvr to infect cells. Sequence analysis of the mutant viruses
revealed three capsid residues that enabled poliovirus to utilize the defective receptors.
Amino acid changes in adapted mutants are located in the VP1 B-C loop at the five-fold
axis, at the interface between protomers near the hydrocarbon binding pocket, and in the
- 29 -
mutations and restore the virus receptor interaction or by affecting receptor contact by
regulating structural transitions the capsid undergoes during receptor interaction (105).
Pvr sequences that contact virus
The binding site for poliovirus appears to be contained within domain 1 (77, 106,
132), which can bind poliovirus when expressed on the cell surface either alone or in a
chimeric molecule fused to CD4 (89), ICAM-1 or Mph. Interaction strength is diminished
when only domain 1 is present on the membrane indicating that domains 2 and 3
participate either directly or indirectly by modulating the structures of domain1.
Mutagenesis of PVR domain 1 has identified the three putative contact regions for
poliovirus: the C-C’ loop through the C’’ strand, the border of the D strand and the DE
loop, and the G strand (3, 16, 105) (Figure 23). Mutagenesis of other loops and strands
has not revealed other regions that are important for binding.
The use of molecular modeling in conjunction with viral genetics revealed that the
C’-C” ridge is likely to be the main part of Pvr that contacts poliovirus. The homologous
part of CD4 plays a major role in the interaction with human immunodeficiency virus
type 1 (82). The D-E loop of domain 1 may also contact poliovirus, but the G strand is
more distant and not likely to be directly involved with the binding site (105). A
chimeric receptor containing the predicted C’C’’D loop-strand region of Pvr was
substituted into the corresponding region of Mph. L cells transformed with the chimeric
receptor became permissive for P1/M but not P2/L and P3/S serotypes of poliovirus. This
indicated that the three serotypes of poliovirus contact Pvr slightly differently (89). These
- 30 -
acid segment, although the contribution of conserved and similar Mph residues cannot be
excluded.
Hydrophobic antiviral agents
Members of the picornavirus family are sensitive to a family of generally
hydrophobic antiviral agents. The drugs include the WIN compounds and a class of
compounds produced by Janssen Pharmaceutica. Crystallographic and cryoEM image
reconstruction studies of virus-drug complexes have shown that these compounds
displace the pocket factor and bind in the hydrophobic core of VP1 (52, 56, 140). Drug
binding has been shown to inhibit infectivity by two different mechanisms. One, drug
binding to virus stabilizes virus, which in turn, inhibits structural transitions necessary for
genome release. The inhibition of transition is caused by an entropic effect (142)
mediated by drug not through increased rigidity of the capsid as has been previously
suggested (100, 140). Second, for some rhinoviruses, drug binding induces limited local
conformational changes in the loops of viral proteins in the canyon; this inhibits receptor
attachment (113, 136). Recently, it has been shown that drug binding interferes with
poliovirus receptor attachment at 4°C but not at physiological temperatures (42). Drug
binding by poliovirus does not result in significant local structural changes at the base of
the canyon (52). These results suggest inhibition of receptor attachment also may be
attributed to the ability of drugs to inhibit small energy-dependent conformational
alterations required for tight receptor binding.
- 31 -
Chapter II. Materials and Methods
- 32 -
Cells and viruses
293-T human epithelial kidney cell line cells were propagated in Dulbecco's
minimal essential medium (Life Technologies, Inc.) containing 10% fetal bovine serum
(HyClone), 100 units of penicillin/ml, and 100 µg of streptomycin/ml (Life Technologies,
Inc.). HeLa cells were propagated in Dulbecco's minimal essential medium containing
10% bovine calf serum, 100 units of penicillin/ml, and 100 µg of streptomycin/ml.
Hybridoma cell line 711C (106) was propagated in HB basal medium plus HB101
supplement (Irvine Scientific). Poliovirus type 1 Mahoney strain, derived from an
infectious cDNA clone (118), was grown in HeLa cells and purified by differential
centrifugation and CsCl density gradient fractionation as described in (124). The ratio of
particles to plaque forming units was determined to be 250:1.
Plasmid construction
Polymerase chain reaction was used to amplify a portion of PVR cDNA that
corresponds to the ectodomain, residues 1-337. DNA encoding 5 histidine residues and a
termination codon were added to the 3'-end during amplification. The 5 histidine residues
were added after a naturally occurring histidine (His-337) in Pvr. The following
oligonucleotide primers were used: 5'-ttgagagacaattgGGAAGCGAGGAGACGCCCG-3'
and 5'-gggagtgacaattgctaatggtggtgatggtgGTGCTCACTGGGAGGTCCCT-3'. Codons for
the additional 5 histidine residues are shown in bold. The Pvr sequence is in capital
letters. The amplified DNA product was inserted into the first cistron position of the
bicistronic vector pCMV/IRES/GFP, resulting in p3DPVR/IRES/GFP/MP8. Expression
of this DNA in mammalian cells should produce a bicistronic mRNA in which the first
- 33 -
(IRES) and the second cistron, which encodes green fluorescent protein (GFP).
Establishment of a Stable Cell Line Expressing sPvr
293-T cells were seeded in 10-cm diameter plastic cell culture plates 1 day before
use. After cells achieved 20% confluence, and 6 h before DNA transfection, the medium
was changed. Ten µg of plasmid p3DPVR/IRES/GFP/MP8 plus 0.1 µg of pRSV-Puro, a
plasmid that contains the puromycin resistance gene, was introduced into 293-T cells by
DNA-calcium phosphate coprecipitation (146). After 18 h of incubation at 37 °C, the
medium was replaced, and incubation was continued for an additional 24 h. For selection
and subculturing of drug-resistant cells, 5 µg of puromycin (Sigma) was added per ml of
medium.
The subpopulation of puromycin-resistant 293-T cells was detached from the
tissue culture plate using cell dissociation buffer (Life Technologies, Inc.), passed
through a 20-µm nylon filter, chilled, and sorted on a Becton Dickinson FACStar with
the excitation wavelength set at 488 nm. A small percentage of the population was
sampled to determine the range of fluorescence intensity. A subpopulation of 1 X 107
GFP-expressing cells with relative fluorescence intensity greater than 95% of the whole
population was collected on ice. GFP-positive cells were cultured for a week, and the
isolation process was repeated with the following modifications. Individual cells with
relative fluorescence intensity greater than 99.75% were clonally isolated and cultured.
Of several cell lines obtained by this procedure, one with the highest level of secretion of
- 34 -
(data not shown).
Protein expression, purification, and modification
At each step of sPvr purification, total protein (Pierce, Inc.), specific activity, and
fold purification were determined. A unit of specific activity was defined as a 20-µl
aliquot capable of neutralizing 50 of 100 plaque-forming units (pfu) in a 100-µl reaction
volume. Fractions containing sPvr were determined by 10% SDS-polyacrylamide gel
electrophoresis and by Western blot analysis (data not shown). sPvr enriched supernatant
was produced by seeding 15-cm diameter plastic cell culture plates to 50% confluency
with GFP-positive cells in 25 ml growth media and subsequent culturing for 10-15 days
without media replacement. Enriched supernatant was mixed with loading buffer A
(LBA, final concentration 50 mM NaPO4, 50 mM NaOH, pH 8, 3 mM imidazole) and
nickel-agarose slurry (final 4% v/v) (Qiagen, Inc.), and incubated at 4 °C overnight with
stirring at 1000 rpm. The slurry was packed into a column (1-cm Bio-Rad Econo-column
with flow adapter) and washed with 10 volumes of wash buffer A (WBA, 10 mM
imidazole in phosphate-buffered saline (PBS), 20 mM NaPO4, 150 mM NaCl, pH 7) at a
flow rate of 1.5 ml/min. Bound protein was eluted with elution buffer A (EBA, 50 mM
imidazole in PBS) at 4 °C at a flow rate of 0.5 ml/min. The sample was dialyzed
overnight against loading buffer B (LBB, 20 mM HEPES-NaOH, pH 8.0, 20 mM NaCl)
prior to application to a Q-Sepharose column (1-cm Bio-Rad Econo column packed with
Q-Sepharose resin charged with counter ions and preequilibrated with loading buffer) at a
flow rate of 0.5 ml/min. After washing with 10 volumes of LBB, at a flow rate of 0.5
- 35 -
in 20 mM HEPES-NaOH, pH 8.0, at a flow rate 0.5 ml/min. One-ml fractions were
collected, and sPvr-containing samples were dialyzed against PBS, and fold purification
was determined.
For the production of deglycosylated sPvr, the GFP-positive clonal population
was expanded and grown for 10 –15 days on 150-cm tissue culture plates that contained
25 ml of growth medium with 100mM deoxymannojirimycin, with repeated pulses of
deoxymannojirimycin every two days for ten days. A Ni agarose enrichment step was
carried out as above. The eluent was dialyzed in the presence of endoglycosidase H in
dialysis/ treatment buffer (D/TB: 10 mM EDTA, 300 NaCl, 20 mM Hepes, 50 mM
Acetate, pH-5.9) overnight at 4°C to allow the reaction to go to completion. The pH of
the reaction was adjusted to 7.5 and the concentration of CaCl and MnCl were adjusted to
5mM and 1mM, respectively. Partially glycosylated sPvr was removed by application to
a double media column: upper layer contained 10 ml G25 media and the bottom
contained 5 ml concanamycin A (Sigma, Inc.), preequilibrated with gel filtration buffer
(GFB: 300 mM NaCl, 5 mM Tris-Cl, pH 7.0, 0.02 % NaN3). The endoglycosidase H was
separated from sdPvr using an S-200 size exclusion column (Pharmacia, Inc.)
preequilibrated with GFB.
The anti-Pvr monoclonal antibody 711C (105) was purified from hybridoma
supernatant using Affi-Gel protein A gel according to the manufacturer's instructions
(Bio-Rad).
- 36 -
Virus neutralization assay and determination of buffers for use with the optical
biosensor
Approximately 200 pfu of poliovirus were incubated with different concentrations
of sPvr in virus dilution buffer (PBS containing 0.02% bovine calf serum) for 30 min at
25 °C followed by 1 h at 37 °C. The virus titer was then determined by plaque assay on
HeLa cell monolayers. To determine if incubation at low pH, the condition used to
remove bound sPvr from virus on the sensor chip, affects virus infectivity, approximately
70 pfu of poliovirus were incubated in 10 mM glycine, pH 3, or PBS, for 5 min, followed
by plaque assay. Plaque assays were carried out essentially as described in (83).
Alteration assays
Preparation of isotopically labeled virus, purification, and alteration assays were
done essentially as reported in (4, 71, 90). For alteration assays, sPvr was incubated with
purified poliovirus in PBS containing 1% bovine serum albumin (Sigma) in a total
volume of 100 µl at 4 °C overnight. The virus-receptor complexes were then shifted from
4 to 37 °C for 0, 5, or 15 min, overlaid onto a 15-30% sucrose density gradient containing
0.1% bovine serum albumin, and centrifuged at 39,000 rpm for 2 h at 4 °C in a Beckman
SW41 rotor. Gradients were fractionated (0.6 ml) from the top to bottom, and
radioactivity was measured in a liquid scintillation counter. In such assays, not all of the
sample can be accounted for, probably due to the hydrophobic nature of the 135 S
particle (46).
- 37 -
Binding of sPvr to poliovirus using an optical biosensor
Surface plasmon resonance experiments were performed on a BIAcore X and
BIAcore 3000 optical biosensor (BIAcore AB) at specified temperatures. Approximately
1,200 response units of purified poliovirus were coupled to flow cell 2 (Fc2) of a CM5
sensor chip via primary amines according to the manufacturer's specifications with the
following modifications. After the activation step, purified poliovirus in PBS was diluted
1:3 with 10 mM sodium acetate, pH 4.5, and injected at 2 µl/min until desired response
units were coupled to the flow cell. The running buffer for the experiments was PBS
containing 0.005% Tween 20 (PBS-T, pH 7.0). For kinetic analysis of the sPvr-poliovirus
interaction, the flow path was set to include both flow cells; the flow rate was 50 µl/min,
and the data collection rate was set to high. Poliovirus was allowed to bind for a 2-min
interval with a wash delay set for an additional 3 min to allow for a smooth dissociation
curve. Settings for equilibrium analyses were the same as for kinetics, except that the
flow rate was set to 2 µl/min. Regeneration of the virus (removal of bound sPvr) was
done by brief pulses of 10 mM glycine at pH 3.0 with or without 300 mM NaCl until the
response was returned to base line. BIAevaluation software, version 3.0, was used to
analyze the surface plasmon resonance data, using global fitting.
- 38 -
Chapter III. Two Distinct Binding Affinities of Poliovirus for
Its Cellular Receptor
With Ann H. Rux, Roselyn J. Eisenberg, and Gary H. Cohen
Department of Microbiology and Center for Oral Health Research, School of Dental
Medicine, and Department of Pathobiology, School of Veterinary Medicine, University
of Pennsylvania, Philadelphia, Pennsylvania 19104
- 39 -
We have examined equilibrium and kinetics of poliovirus binding to sPvr. In the
study of virus entry, determination of biochemical and biophysical parameters of virus
receptor interactions are important because these parameters describe the dynamics of the
interaction. In addition these parameters enable a better understanding of the reaction in
comparison to other systems. The results of such studies, together with structural and
genetic analyses of the virus-receptor interaction, provide a complete picture of early
events in infection (61, 80, 125, 153). To study the kinetics and equilibrium of poliovirus
binding to Pvr, we used surface plasmon resonance (23, 73) to examine the interaction of
a soluble form of Pvr (sPvr) with poliovirus. sPvr expressed in and purified from
mammalian cells is able to bind poliovirus, neutralize viral infectivity, and induce the
formation of altered particles.
Expression and purification of sPvr in mammalian cells
A novel approach was used to express a soluble form of the poliovirus receptor at
high levels in mammalian cells for biochemical and biophysical studies. A plasmid was
used that leads to the production of a bicistronic mRNA upon expression in mammalian
cells. The coding region of the Pvr ectodomain (with a 6-histidine tag at the C terminus,
Figure 6) was placed in the first cistron position, followed by an IRES, and then the
coding region for GFP in the second cistron. In a cell line stably expressing the
bicistronic mRNA, the intensity of GFP fluorescence is an approximate indicator of the
expression of the protein in the first cistron. Fluorescence-activated cell sorter analysis
was then used to isolate a clonal cell line that contains a fluorescence intensity greater
than 99.75% of the GFP-positive population.
- 40 -
mg/liter sPvr. sPvr was purified from cultured supernatant using a two-step procedure.
The level of purification was determined by assaying the capacity of sPvr to neutralize
infectious poliovirus (71). In the first step, nickel affinity chromatography achieved 160fold purification over the cultured supernatant (Table 1). In the second step, Q-Sepharose
purification ion exchange chromatography achieved 2.3-fold purification over the
previous step. At this stage of purification, sPvr was the only visible band on a
Coomassie Blue-stained, SDS-polyacrylamide gel (Figure 6). Edman degradation
revealed that the N terminus of purified sPvr begins at Asp-28 of the unprocessed
precursor, as previously reported for the membrane-bound form (17). Although the
predicted molecular mass of sPvr is 34 kDa, the purified protein migrates as a diffuse
band between the 61- and 85-kDa molecular mass markers (Figure 6), suggesting that the
protein is heavily glycosylated. After treatment of sPvr with N-glycosidase F, which
cleaves asparagine-linked glycan chains on glycoproteins, the polypeptide migrates as a
smear at 34 kDa, the predicted size of the non-glycosylated protein (see deglycosylated
sPvr section). A similar protein produced in insect cells migrated at 51 kDa, probably due
to less extensive glycosylation in that cell type (4).
Virus neutralization and alteration activity of sPvr
We carried out several assays to determine whether purified sPvr is biologically
active. Plaque reduction assays were used to determine the efficiency of neutralization of
poliovirus by sPvr. Viral infectivity was reduced by 50% at 30 nM sPvr (Figure 7). In
- 41 -
Figure 6: Purity of recombinant sPvr expressed in mammalian cells. Left, schematic
diagram of sPvr, indicating the first and last amino acids of the recombinant protein. An
additional 5 histidine residues were added to the C terminus of the protein. Potential Nlinked glycosylation sites are designated by a ball and stick. Disulfide bonds are indicated
by SS. Right, SDS-polyacrylamide gel electrophoretic analysis of purified sPvr. Lane 1,
1.1 µg; lane 2, 0.6 µg; lane 3, molecular mass markers.
- 42 -
Table 1: Purification of sPvr
Procedure
Enriched cell culture
supernatant
Total
protein
Total
activity
Recovery
Specific
activity
Purification
mg
unitsa
%
units/mg
-fold
1,330
50,000
38
Nickel-agarose
3.7
22,000
45
6,000
Q-Sepharose
0.65
9,000
40
14,000
a
1 unit is a 10 µl aliquot capable of neutralizing 50 of 100 pfu.
- 43 -
160
2.3
contrast, the 50% inhibitory dose for infectivity (IC50) of sICAM-1 for rhinovirus type 3
was 10-fold higher than sPvr, 300 nM (97).One possible explanation for this difference is
that the affinity of poliovirus type 1 for its soluble receptor is greater than that of
rhinovirus 3 for sICAM-1 (see below).
We also determined if sPvr is capable of inducing structural changes in
poliovirus. This question was addressed by incubating sPvr with poliovirus at 37 °C and
assaying the products by sucrose gradient centrifugation. At the concentration of sPvr
used, 1.8 X 10-8 M, conversion of native virus (160S) to 135S altered particles and 80 S
empty capsids was nearly complete within 15 min (Figure 8). These results indicate that
sPvr produced in mammalian cells can efficiently bind to poliovirus and induce the
structural changes associated with cell entry.
Conditions and specificity of surface plasmon resonance
Surface plasmon resonance allows determination of quantitative affinities (KD),
association (ka), and dissociation (kd) rates for the formation and dissociation of the virusreceptor complex (28, 30, 84, 128). To examine the kinetics of binding of sPvr to
poliovirus by surface plasmon resonance, purified poliovirus was coupled to the sensor
chip surface, and sPvr was injected over the chip surface. An example of raw sensorgram
data is shown in Figure 9. In this experiment, flow cell 2 contained immobilized
poliovirus, and flow cell 1 was activated and blocked without virus. sPvr was injected,
and its association with virus was followed for 2 min. At 120 s, sPvr was replaced with
buffer, and the dissociation of complex was followed for 3 min. The response on
- 44 -
Figure 7: Neutralization of poliovirus infectivity by sPvr. Approximately 200 pfu of
poliovirus were incubated with different concentrations of sPvr at 37 °C for 1 h.
Remaining infectivity was determined by plaque assay on HeLa cell monolayers. The
percent reduction of pfu was calculated relative to virus incubated with buffer only. The
means ± S.D. of three experiments are shown. The IC50 of sPvr was extrapolated from a
line graph of the same data.
- 45 -
Figure 8: Kinetics of sPvr-induced conformational changes of poliovirus. [35S]
Methionine-labeled poliovirus (5 X 109 virions) was incubated with sPvr (1.6 X 10-8 M)
at 4 °C overnight, then shifted to 37 °C for 0 (squares), 5 (triangles), or 15 (circles) min.
Samples were centrifuged in a 15-30% sucrose gradient. Untreated 160 S particles and 80
S particles obtained by heating 160 S particles for 20 min at 56 °C were centrifuged in a
parallel gradient as markers.
- 46 -
the y-axis is measured in response units. The sensorgram reveals a change in the bulk
refractive index, but there was no significant background response when 1.3 µM sPvr
was injected over the mock-coupled control surface. In the surface plasmon resonance
experiments that followed, data from flow cell 2 were subtracted from the data from flow
cell 1 to correct for changes in bulk refractive index. These results demonstrate binding
of sPvr to poliovirus immobilized on the chip surface.
The sensor chip surface was regenerated by treatment with low pH, to disrupt the
virus-receptor interaction. Poliovirus remaining on the chip surface should survive these
conditions, because its natural route of infection is through the acidic environment of the
stomach. Two experiments were done to ensure that the sensor chips could be reused.
First, unbound poliovirus was incubated in regeneration buffer (glycine buffer, pH 3) for
5 min at room temperature, and then infectivity was determined by plaque assay. As
expected, this treatment did not reduce poliovirus infectivity, suggesting that conditions
used for regeneration of the sensor chip do not disrupt virus structure (Figure 10).
Second, repeated use and regeneration of sensor chips containing bound poliovirus did
not affect sensorgrams and response levels (data not shown).
To determine the specificity of the poliovirus-sPvr interaction, a blocking
experiment was performed using a monoclonal antibody, 711C, directed against the first
domain of Pvr and which prevents poliovirus attachment to cells (105). Two
concentrations of monoclonal antibody 711C were preincubated with sPvr for 1 h at 4 °C
prior to injection onto the sensor chip containing bound poliovirus. Preincubation with
- 47 -
Figure 9. Example of raw sensorgram data. sPvr (1.3 µM) was injected over a mockcoupled surface (blue line, flow cell 1) in series with a surface containing 1360 response
units (RU) of poliovirus (green line, flow cell 2). At 120 s, the sample was replaced with
buffer, and dissociation was followed for 2 min. The flow cell 2-flow cell 1 sensorgram
(red line) represents the data corrected for changes in bulk refractive index and
nonspecific binding of sPvr to the chip surface.
- 48 -
11). A control monoclonal antibody DL11, directed against herpes simplex virus
glycoprotein D, did not inhibit the formation of the poliovirus-sPvr complex (data not
shown). These results indicate that the sPvr-poliovirus interaction under study resembles
the interaction during infection of cells, since it is mediated by domain 1 of sPvr.
Kinetic and equilibrium affinity analysis
Determination of kinetic binding parameters for the sPvr-poliovirus interaction was done
at 20 °C using separate injections of 2.5-fold serial dilutions of sPvr onto the sensor chip
containing bound poliovirus. The sensorgrams of the sPvr-poliovirus interaction were
imposed upon different model curves generated by global fitting analysis (Figure 12).
The data fit best with the parallel reactions (2 sites) model, A + Β1 ⇔ AB1, A + B2 ⇔
AB2. The X2 values generated using this model for interaction at 20 °C were below 1.5,
indicating an excellent fit. On the other hand, the X2 value for a one-site binding model
was 29, demonstrating a poor fit to that model. The two affinity constants calculated
from the surface plasmon resonance data are 0.67 µM (KD1) and 0.11 µM (KD2) (Table 2).
The calculated association rate constants are 3.6 X 103 M-1 s-1 (ka1) and 3.2 X 104 M-1 s-1
(ka2); the dissociation rate constants are 2.4 X 103 s-1 (kd1) and 3.3 X 103 s-1 (kd2) (Table 2).
Binding rates were unaffected by changes in flow rate, demonstrating that the poliovirussPvr interaction is not limited by mass transport (data not shown) (72). The kinetics and
affinity analysis of the rhinovirus-sICAM interaction using the biosensor, as well as
affinity analysis in solution, was also shown to be biphasic (28). In that study, the linear
transformation method was used to analyze biosensor data on the rhinovirus-sICAM-1
- 49 -
Figure 10:
Effect of low pH treatment on poliovirus infectivity. Approximately 70
pfu of poliovirus were incubated in 10 mM glycine, pH 3, or PBS, for 5 min, followed by
plaque assay. Shown is the average of two experiments.
- 50 -
Figure 11:
Specificity of sPvr interaction with poliovirus on sensor chip surface.
Anti-Pvr monoclonal antibody 711C was preincubated with sPvr (4 µM) for 1 h at 4 °C
prior to injection onto the sensor chip containing bound poliovirus. At 180 s, the sample
was replaced with buffer, and dissociation followed for 3 min. red line, no 711C; blue
line, 2 µM 711C; green line, 4 µM 711C.
- 51 -
also yields biphasic plots indicative of two binding sites (data not shown).
Binding of sPvr to the sensor chip was repeated under equilibrium conditions to
confirm the existence of two classes of binding sites, and the affinity constants were
determined by Scatchard analysis (129). The contact time was varied from 50 min for the
lowest concentration to 10 min for the highest concentration of sPvr (Fig. 13A). The
Scatchard plot of the equilibrium data is curved, indicating that there are two classes of
sPvr-binding sites on poliovirus at 20 °C, with binding affinities of 1.1 µM (KD1) and
0.16 µM (KD2) (Fig. 13B) . These values are similar to those obtained by kinetic analysis
(Table 2).
To determine the effect of temperature on the poliovirus-receptor interaction, the
kinetics experiments were repeated at 5, 10, 15, and 20 °C. Higher temperatures, at which
receptor-induced virus disruption occurs, were not studied because it would be difficult to
interpret the biosensor data (25). With increasing temperature, the value for KD1
decreased, indicating a rise in affinity (Table 3). Binding of sPvr at these sites on
poliovirus is therefore endothermic. The value for KD2 did not exhibit a general increase
or decrease with temperature, and therefore the thermodynamic nature of this site could
not be determined.
The relative abundance of the KD1 and KD2 sites at different temperatures was
calculated from the kinetics data using global analysis software, assuming a parallel
reactions model. At 20 °C, the KD2 site constituted approximately 46% of the total
binding sites on the sensor chip (Table 3, %Rmax2). The relative abundance of the KD2 site
decreased with decreasing temperature. At 5 °C, the relative abundance of the KD2 site is
- 52 -
Figure 12: Corrected sensorgram overlays for the interaction of decreasing
concentrations of sPvr with immobilized poliovirus. Data were collected at 5 Hz.
Concentrations of sPvr: red line, 8 µM; blue line, 3.2 µM; green line, 1.3 µM; magenta
line, 0.51 µM; turquoise line, 0.21 µM. The black lines are the best global fit to the
parallel reactions model (BIAevaluation 3.0 software).
- 53 -
Table 2
Kinetic and affinity parameters for sPvr binding to poliovirus type 1 at
20 °C
Kinetic constants were measured as described in Figure 12 for binding of sPvr
to poliovirus type 1.
ka1
ka2
kd1 x10-3 kd2 x 10-3
KD1a
KD2
M-1 s-1
s-1
3,600 ± 660b 32,000 ± 2,600 2.4 ± 0.9 3.3 ± 0.4
a
b
KD = kd/ka.
Mean of three experiments ± S.D
- 54 -
µM
0.67 ± 0.28 0.11 ± 0.02
D1
constant at all temperatures tested.
- 55 -
Figure 13:
Equilibrium binding sensorgrams and Scatchard analysis of the
binding of sPvr to immobilized poliovirus. A, binding of sPvr to immobilized
poliovirus was monitored for 10 min for the injections of 13.5 (red line), 9 (blue line), 6
(green line), 4 (magenta line), and 2.67 (turquoise line) µM concentrations; 20 min for
injections of 1.78 (gold line), 1.18 (black line), 0.79 (yellow line), 0.53 (pale blue line),
and 0.35 (pink line) µM concentrations; and 50 min for injection of the 0.23 µM (salmon
line) concentration. Arrows indicate the time points used for the Scatchard analysis. B,
Scatchard analysis. C is the concentration of sPvr flowed across the sensor chip surface at
20 °C. The negative slope of each line is equal to each association constant; the
reciprocals are the KD values. The R2 values for the linear fit of the data were 0.95 and
0.91 for KD1 and KD2, respectively.
- 56 -
- 57 -
Table 3
Affinity constants for sPvr binding to poliovirus type 1 and abundance of
each binding class at different temperatures.
Temperature
KD1
KD2
Rmax1
Rmax2
%Rmax2a
°C
5
µM
1.56c
RUb
0.006
75 (4)
10 (6)
12
10
1.30
0.166
86 (3)
28 (8)
25
15
1.00
0.096
85 (0.3)
45 (2)
35
20
0.67
0.11
71 (0.6)
61 (3)
46
a
Rmax2/(Rmax1 + Rmax2) = 100. Rmax is the maximum receptor binding capacity,
and therefore %Rmax2 is the relative abundance of the binding site that
corresponds to KD2.
b
Resonance units, calculated with BIA evaluation 3.0, assuming a parallel
reactions model. Mean of two experiments and range in parentheses.
c
Mean of two experiments.
- 58 -
Chapter IV. Three-dimensional structure of poliovirus
receptor bound to poliovirus
With David M. Belnap*, David J. Filman †, Naiqian Cheng*, Benes L. Trus*,§
Harmon J. Zuccola †, James M. Hogle †, and Alasdair C. Steven*
* Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal and
Skin Diseases, Bethesda, MD 20892; † Department of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; and
§ Computational Bioscience and Engineering Laboratory, Center for Information
Technology, National Institutes of Health, Bethesda, MD 20892
- 59 -
Multiple genetic approaches have been used to study the poliovirus-Pvr
interaction. Poliovirus mutants have been selected for resistance to neutralization (33)
with a solubilized form of Pvr or for the ability to utilize mutant Pvr (35, 88, 89, 105).
Other virus mutants were generated by site-directed mutagenesis in the virus capsid (57).
Analysis of these mutants suggests that the principal contact site of Pvr on the capsid is
the floor of the canyon, above the hydrocarbon-binding pocket, and on the outer ("south")
rim of the canyon. Additional sites that modulate receptor utilization are located at or
near the peak of the mesa. Mutagenesis of Pvr DNA has revealed that the binding site for
poliovirus is contained in domain 1 of Pvr (d1), the membrane-distal domain (77, 106,
132). Mutations in the predicted C'C", CC', DE, and EF loops, and the C"- and Dstrands of this Ig-like domain disrupt virus binding (3, 16, 105).
To complement these genetic analyses, we have analyzed the complex of
poliovirus type 1 bound to sPvr by cryoelectron microscopy. The resulting reconstruction
revealed the manner in which domain 1 of Pvr inserts into the canyon. It also helped
guide construction of a homology model of Pvr. This model, together with the known
crystal structure of poliovirus (63), was used to identify specific interactions between the
virus and receptor.
Visualization of poliovirus decorated with sPvr
Cryo EM image reconstructions were generated in the following manner. Briefly,
the conditions used for binding sPvr to poliovirus were short incubations at 4°C to avoid
converting virions to 135S particles, which occurs at higher temperatures (4, 51). The
- 60 -
micrographs were then processed into image reconstructions.
With or without the addition of sPvr, the most prominent feature in
cryomicrographs of poliovirus is its roundish shape (Figure 14a). Faint protrusions are
discernible on labeled virions and represent attached sPvr molecules. The bound
receptors are much more obvious after image reconstruction (see Figure 14b-d), which
shows prominent protrusions. The protrusions are 115 Å long, 20-60 Å wide, and extend
outwards at an oblique angle relative to the capsid surface. The comparison of
corresponding cross sections through the virion-sPvr and virion density maps shows that
the capsid is largely unchanged on binding receptor under these conditions (13). The
sPvr-bound virion does not show the redistribution of RNA density characteristic of the
135S particle (12). However, a subtle change in the poliovirus capsid was observed in the
reconstructions: a small tunnel beneath the receptor-binding site on the floor of the
canyon that extends into the "pocket-factor" binding site in VP1 was present (Figure 15b)
(58). This result indicates that the pocket factor is possibly expelled during Pvr binding.
The structure of the receptor fragment
A representation of sPvr from the difference map could be constructed because
there is structural invariance of the virion on receptor binding (Figure 14e). The three Iglike domains are obvious in the reconstructions. We equate them with domains d1, d2,
and d3 (102), in order of increasing distance from the capsid surface. d1 is contacted by
the poliovirus capsid. d2 has two prominent nodules and is connected to d1 by a narrow
region. The long axes of these two domains are aligned, and they appear to be firmly
- 61 -
Figure 14:
CryoEM of poliovirus labeled with sPvr. (a) Cryomicrographs of
poliovirus particles complexed with (Top) and without (Bottom) sPvr. Bar = 300 Å.
Image reconstructions are shown of virion + sPvr [in stereo (c)] and, for comparison, of
the virion (b) (from (12)). The two reconstructions were overlaid in d with the respective
contour levels adjusted to clarify the interaction of sPvr with the virion. Bar = 100 Å. (e)
Two views of a single sPvr molecule extracted from the difference map (13). Domain
boundaries are marked. Bar = 25 Å.
- 62 -
Figure 15:
Analysis of the Reconstructions (a) "Road map" representations of
poliovirus (Left) and rhinovirus-14 (Right). The corresponding triangular area of the
capsid surface, bounded by a 5-fold and two 3-fold icosahedral symmetry axes, is marked
(Inset). The radial distances of surface residues from the virion center are color coded and
contoured [see key (Top Right)]. The receptor footprints are shown in white. (b) A ribbon
diagram of the sPvr model is flanked by two views (68) of a single sPvr molecule as
portrayed in the cryoelectron microscopy density map (white cage), enclosing the model
of the three sPvr domains, d1 (cyan), d2 (orange), and d3 (violet). Carbohydrates attached
to d2 [to N188 (Left) and N237 (Right)] and possibly to d1 are shown in brown. Also
shown are the capsid proteins VP1 (blue), VP2 (yellow), VP3 (red), and VP4 (green).
The tunnel beneath the sPvr-binding site is evident (white arrows). "Pocket factor" is
magenta. (c) The sPvr sequence is mapped onto secondary structural elements of the
homology model. Asn residues thought to be glycosylated are marked with asterisks. (d)
Ribbon diagram showing the docking of the sPvr model onto the capsid surface. Same
color conventions as in b. The axes allow this view to be related to Figure 23. (e)
Schematic diagram showing a possible binding configuration of poliovirus with intact
membrane-bound Pvr.
- 63 -
- 64 -
between its long axis and that of d2. Its density is lower, suggesting that d2 and d3 may
be more loosely joined (13).
Contact area on poliovirus
The receptor fragment binds at a glancing angle, such that, its d1 domain extends
into the canyon and makes contact with the capsid surface near the center of the
icosahedral asymmetric unit bounded by a 5-fold axis and two 3-fold axes (Figure 15a).
The receptor appears to bridge the canyon with major contact points are localized in a
cleft on the "south rim” of the canyon and on the side of the mesa on the "north rim,"
(Figures 14c, d, and 15a). The area of contact of sPvr to poliovirus differs substantially
from that of the rhinovirus receptor, intercellular adhesion molecule-1, to rhinovirus
(111).
Footprints of the interaction were created by overlaying the virion-sPvr
reconstruction and the difference map on the atomic-resolution coordinates of the virion,
we found that the sPvr-binding site includes many residues in VP1 (102-108, 166-169,
213-214, 222-236, 293-297, 301-302), a few in VP2 (140-144, 170-172), and several in
VP3 (58-62,93, 182-186). The footprint of sPvr on the capsid surface consists of three
distinct patches. Two of the patches are similar to the footprint of intercellular adhesion
molecule-1 on rhinovirus (111) (Figure 15a). The third patch in the southeast corner is
unique.
- 65 -
The homology models for d1, d2, and d3 were fitted into the reconstruction
(Figure 15b). The density map exhibits constrictions between the domains. Consequently,
determining the placement of the domains was mainly a matter of fixing their orientations
about their long axes. The d1 model could be fitted into the density map in either of two
orientations, 180o apart. One orientation seemed more consistent with mutational data
implicating the C'C" and DE loops of Pvr, and the EF (166-169) and GH (213-236)
loops of VP1, the EF loop of VP2 (140-144), and the GH loop of VP3 (182-186) as
important interaction sites (Figure 23); the other orientation was less consistent with these
data. The orientations of d2 and d3 were unambiguous. β-strand and loop assignments in
the final model (Figure 15b Inset) are given in Figure 15c. The relative orientation of d1
and d2 in this pseudoatomic model is controversial because Pvr would be the only
example of the V and C-like domains in this orientation; all other known examples of the
Ig-superfamily structures are in the reverse orientation by 180° (J. Hogle, personal
communication). However, the presented model of Pvr agrees with most of the
mutational data and fits well into the density cage generated by the reconstructions. This
controversy remains to be resolved.
The pseudo atomic model of domain 1 (d1) model (residues 29-142) fits the
reconstructed density well and exhibits notable complementarity with the virus surface.
Adaptation of the initial homology model to fit the density map required major changes
in only one large loop (CC'). This loop, which projects laterally from the side and top of
the β-sandwich in all close-sequence homologues, would protrude significantly through
the envelope in any plausible orientation of d1 unless the loop were folded closer to the
- 66 -
C-strands of VP1 and the FG loop of the receptor as presently modeled, suggesting that
the FG loop may adopt a different conformation than in the homologues. There is some
extra density between the receptor and the virus surface in the vicinity of N105, which is
a potential glycosylation site (Fig. 15 b-d); but, this feature appears too small to
accommodate a full-length glycosyl modification. Interestingly, the mutant N105A,
which is not glycosylatable at this site, exhibits increased virus binding and infectivity
(16). There is no evidence for glycosylation at N120.
In the pseudo atomic model of domain 2 (d2-residues 143-242) the orientation
was constrained by three factors. Firstly, two of the glycosylation sites should coincide
with the prominent nodules that extend laterally on either side of the domain. Secondly,
the convex shape of one face and concave shape of the opposite face of the domain
should match between the density map and the model (see Fig. 15b). Lastly, the C
terminus of the G-strand should be located near d3. In the preferred orientation, the
glycosylation sites at N188 and N237 account for the lateral protrusions. The close
proximity of N218 to N237 makes it difficult to determine whether N218 is glycosylated
as well.
The d3 model (residues 243-330) fits its portion of the envelope well (Fig. 15b),
despite the weakness of this density compared with d2 and d1. The orientation of d3 is
constrained by the requirements to position its A-strand on the lower surface of d3, and to
fit the large CD loop into a bulge on the top surface of the envelope. Thus positioned, the
d3 model is clearly shaped similarly to its envelope (Fig. 15b). There is no compelling
evidence for glycosylation at any of d3's three potential sites.
- 67 -
Deglycosylated sPvr (sdPvr)
To resolve the controversy between our results and the results of others (58, 149)
that demonstrate a reverse orientation of domain 1 of sPvr in the canyon of poliovirus, we
produced a deglycosylated soluble form of Pvr (sdPvr). This may resolve the controversy
because glycosylation of N105 on the receptor at the sPvr interface between virus and
receptor may add extra density to the vicinity; these sugar moieties make modeling the
receptor and its interaction difficult. Removal of all N linked sugar residues may
improve the model because only amino acids would be present at the site of interaction.
sdPvr was produced by expressing sPvr with mainly high mannose glycosylation
linkages, in mammalian cells, using 1-deoxymannojirimycin and ensuing removal of high
mannose glycosylation linkages with endoglycosidase H treatment (Figures 16 & 17).
sdPvr possessed biological function in agreement with previously published results that
suggested glycosylation of domain 1 of membrane bound Pvr decreased virus binding
(16). Virus binding was significantly enhanced when membrane bound Pvr lacked
carbohydrate chains in domain 1. sdPvr neutralized poliovirus at lower concentrations
than sPvr in the neutralization assay, with IC50 values of 0.0055 and 0.021µM,
respectively (Figure 18). The initial cryoEM image reconstruction, to 25 Å, of sdPvr
complexed with poliovirus (Figure 19, left), demonstrated that the interacting receptor
has the same projection geometry from the virus as the sPvr interaction (Figure 19, right).
As predicted, the "arms" of domain 2 are gone in the sdPvr structure. Therefore, the
“arms” are likely to be formed by glycosylation. Domain 1 of sdPvr may be smaller than
sPvr. A reasonable explanation for this observation is that the majority of the
- 68 -
The reconstructions using sdPvr are in agreement with the enhanced neutralization
capacity of sdPvr. The reason the results seem to be in agreement is because the
glycosylation that causes steric hindrance at the site of interaction is not present.
Recently, the naturally occurring form of soluble poliovirus receptor, sCD155,
was purified from human serum to ~10% purity by immunoaffinity purification and
provided to us by Denis group. It is not clear the relative proportion of H 20 A∆1 and H
20 A∆2 soluble receptor variants present in this preparation (Figure3). A comparison of
the activities of sPvr and sCD155 was made to determine if recombinant sPvr is
functioning similarly to naturally occurring receptor. A side-by-side comparison of sPvr
and sCD155 by neutralization assay demonstrated that both proteins yield similar
neutralization profiles, indicating that both proteins are functional at similar
concentrations (Figure 18). This result lends strong evidence to the notion that
recombinant sPvr is functioning in a manner similar to Pvr purified from human serum.
Since sCD155 is only ~10% pure, an accurate IC50 could not be determined.
The availability of pure and quantifiable sPvr allowed us to reexamine srr
mutants; we determined if, and to what degree, each variant was resistant to sPvr. This
study is important because, in conjunction with thermodynamic and kinetic studies of the
mutants, it will allow for determination of the mechanism of resistance for the mutants.
srr mutants differ in several parameters: location and type of residue substitution on
capsid; affinity for cell surface Pvr; and capacity to undergo alteration when contacting
membrane bound Pvr (Table 4, Figure 22). Many of the srr mutants predicted the site of
receptor contact to be in the canyon of the virus. To initiate studies that will allow us to
- 69 -
Figure 16:
Trimming of an asparagine-linked precursor to a high-
mannose structure.
High-mannose structures are formed first during the processing reactions, by
exoglycosidase trimming of the precursor as outlined above. In the endoplasmic
reticulum, Glucosidase I initiates the trimming reactions, removing the first glucose
molecule from the precursor (structure I) to form structure II. Structure II is converted to
structure III (cleavage of bonds 2 and 3) by glucosidase II. An endoplasmic reticulum
α1,2-specific mannosidase is responsible for the next step, removal of a single mannose
in the middle branch (bond 6, structure II) of the precursor. M8GN2-Asn (structure IV) is
transported from the endoplasmic reticulum to the Golgi apparatus. There, the Golgi αmannosidase I (also α1,2-specific) trims three more mannose residues (cleavage of bonds
4, 5, 7) from the precursor. Further oligosaccharide processing may occur in the Golgi
apparatus to form complex and hybrid oligosaccharides.
1-deoxymannojirimycin blocks Golgi α-mannosidase I; this stops the processing
of N-linked high-mannose oligosaccharides to complex oligosaccharides. Treatment of
soluble receptors containing high mannose linkages with endoglycosidase H usually
results in the removal of all N-linked sugar residues but the peptide proximal Nacetylglucosamine.
- 70 -
- 71 -
1
2
70 kD
30 kD
Figure 17: Purity of processed recombinant sdPvr expressed in mammalian
cells. SDS-polyacrylamide gel electrophoretic analysis of purified sdPvr. Lane 1 and 2
are 4 µl aliquots of from fractions eluted from the S-200 gel filtration column. Right of
the Coomassie blue stained gel are the positions of molecular mass standards.
- 72 -
Neutralization of poliovirus by sdPvr
and sPvr
100
sPvr
sdPvr
50
0
Concentration Pvr (uM)
Figure 18:
Neutralization of poliovirus infectivity by sdPvr and sPvr.
Approximately 200 pfu of poliovirus were incubated with different concentrations of
sdPvr or sPvr at 37 °C for 1 h. Remaining infectivity was determined by plaque assay on
HeLa cell monolayers. The percent remaining pfu was calculated relative to virus
incubated with buffer only. The IC50 of sPvr and sdPvr were extrapolated from a line
graph of the same data.
- 73 -
Figure 19:
CryoEM of poliovirus labeled with sdPvr. Image reconstructions are
shown of virion + sdPvr (left) and virion + sPvr (right) for comparison. The yellow arrow
denotes a glycosylation ‘arm’ of sPvr. Orange arrow denotes decreased density of sdPvr
at site of interaction; Pink arrow shows similar site involving sPvr + virion.
- 74 -
Neutralization of poliovirus with
sCD155 and sPvr
100
sPvr
sCD155
50
0
Concentration of receptor (uM)
Figure 20:
Neutralization of poliovirus infectivity by sCD155 and sPvr.
Approximately 200 pfu of poliovirus were incubated with different concentrations of s
CD155 or sPvr at 37 °C for 1 h. Remaining infectivity was determined by plaque assay
on HeLa cell monolayers. The percent remaining pfu was calculated relative to virus
incubated with buffer only.
- 75 -
Neutralization of srr mutants by sPvr
111c
312
612
1022
211B
1072
151A
R252.0
382
251A
332
212
242
292
P1/M
100
75
50
25
0
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Concentration of sPvr, uM
B.
Mutant
IC50
µM
111c
ND
312
0.27
Figure 21:
612
0.21
1022
0.20
211b
0.18
1072
0.17
151a
0.079
R252
0.069
382
0.063
251a
0.056
332
0.053
212
0.041
242
0.020
292
0.010
A. Neutralization of poliovirus wt. and srr mutants by sPvr.
Approximately 200 pfu of poliovirus were incubated with different concentrations of
sPvr at 37 °C for 1 h. Remaining infectivity was determined by plaque assay on HeLa
cell monolayers. The percent pfu remaining was calculated relative to virus incubated
with buffer only. The IC50 of sPvr was extrapolated from a line graph of the same data.
B. Table of 50%inhibitory concentrations for wt and srr viral variants.
- 76 -
P1/M
0.0061
50
for each variant. The results indicate that there is a significant difference in the level of
resistance to neutralization by sPvr between the variants. The level of resistance ranges
from 0.0061 µM for wt poliovirus to greater than 0.27 µM for the mutant 111c (Figure17,
Table 4).
- 77 -
Chapter VI. Discussion
- 78 -
Kinetics of poliovirus interaction with sPvr
To measure kinetic constants of the poliovirus-receptor interaction, we expressed
and purified from mammalian cells a soluble form of the poliovirus receptor. Surface
plasmon resonance was used to study binding of poliovirus with sPvr. The affinities
determined by biosensor are within 1 order of magnitude of the IC50 of sPvr determined
by plaque assay, suggesting that the values determined by BIAcore could be the
functional affinities for sPvr.
The results indicate that the interaction between poliovirus and sPvr is biphasic.
Two classes of binding site for sPvr on poliovirus were detected called the KD1 site and
the KD2 site. At 5 °C, approximately 90% of the binding sites were KD1 sites, with a
binding affinity of 1.56 µM. The fraction of KD2 sites, with a binding affinity of 0.11 µM,
increases with temperature and constitutes 50% of the sites at 20 °C. A biphasic binding
model for poliovirus and Pvr has not been described previously. The binding affinity of
poliovirus for the surface of HeLa cells was previously determined to be approximately
10-10 M at 4 °C (19, 34). We find that the binding affinity of the KD1 site, the predominant
binding site at this temperature, is 4 orders of magnitude lower. The difference may be
explained by the fact that the binding affinities calculated in the present study represent
the intrinsic affinity of poliovirus for a single receptor molecule. In contrast, receptor
molecules may cluster on the cell surface, increasing the apparent affinity, or avidity, of
the virus-receptor interaction. Such clustering does not occur in solution (97). In another
study, a single binding affinity of poliovirus for a soluble form of Pvr produced in insect
cells was determined to be 4.5 X 10-8 M at 4 °C (4). In those studies, binding assays were
- 79 -
the KD2 site, it is not clear why the lower affinity site was not detected. One possibility is
that concentrations of sPvr were not sufficiently high to detect the lower affinity site. In
addition, proteins produced in insect cells and in mammalian cells have different patterns
of glycosylation, which might contribute to the different results. An N-linked
glycosylation site within Pvr domain 1 is known to influence its interactions with
poliovirus (16) and may contact the receptor binding site on the viral capsid (13). A sideby-side comparison must be done to resolve this issue.
The finding of two classes of receptor-binding sites on a virus has also been
reported for rhinovirus type 3 and a soluble form of its cellular receptor, ICAM-1 (27,
28). Although the rhinovirus-sICAM and poliovirus-sPvr interactions are biphasic, there
are significant differences in the affinity and kinetic constants. The association rates ka1
and ka2 are 25- and 13-fold higher for the poliovirus-sPvr interaction than for the
rhinovirus-sICAM interaction at 20 °C. The greater association rate of poliovirus-sPvr
might be due, in part, to differences in the extent of contact between virus and receptor.
Three-dimensional models of virus-receptor complexes produced from cryo-electron
microscopy and image reconstruction reveal that the footprint of Pvr on poliovirus is
significantly larger than that of ICAM-1 on rhinovirus (13, 58, 79, 149). The extra
surface area on poliovirus includes the knob of VP3 and the C terminus of VP1 from the
5-fold related promoter in the southeast corner of the road map describing the contact of
Pvr on poliovirus (13). In contrast, although there are two dissociation rate constants for
poliovirus-sPvr, only one has been reported for the rhinovirus 3-sICAM interaction (27,
28). The dissociation rates for the poliovirus-sPvr interaction are 1.5- and 2.0-fold faster
- 80 -
complex. The affinity constants for the poliovirus-sPvr interaction are 19- and 6-fold
greater than those reported for the rhinovirus·sICAM-1 complex (28). Consistent with
these differences is the fact that the IC50 of sICAM-1 for rhinovirus 3 is 10-fold higher
than that of poliovirus (97). However, other factors might play a role, including the
number of receptors per virus particle that are required to neutralize infectivity.
The effect of temperature on the interaction of poliovirus with sPvr was studied.
Binding at the lower affinity site, KD1, is endothermic (e.g. heat is absorbed by the
complex), similar to both sites on rhinovirus (27, 28). As suggested previously, heat
absorbed during the interaction of virus with receptor might help to lower the energy
barrier required for uncoating of the virus particle (28).
In contrast to the observations with poliovirus and rhinovirus, a single class of
binding site (KD = 3.0 X 10-6 M at 20 °C) was found on echovirus 11 for a soluble form
of its receptor, CD55 (84). The affinity of this interaction is at least 4 times lower than
either of the binding sites on poliovirus for sPvr. Like most protein-protein interactions,
the affinity of echovirus 11 for CD55 increases with decreased temperature, indicating
that binding is exothermic. The association rate for the interaction between echovirus 11
and CD55 is faster than that of poliovirus-sPvr (39- and 4.4-fold) and rhinovirus-sICAM1 (28). One explanation for these findings is that the contact between echovirus 11 and
CD55 is more extensive than that of the other two virus-receptor complexes. In addition,
the binding site for CD55 on echovirus 11 might be more accessible than those of Pvr and
ICAM-1, which are located in a depression on the capsid (13, 58, 149). The dissociation
- 81 -
poliovirus-sPvr or rhinovirus-sICAM-1 (28). These findings are consistent with a more
accessible binding site for CD55 on echovirus 11, compared with the receptor-binding
sites on poliovirus and rhinovirus (28, 84). In addition, it is possible that the atomic
interactions between CD55 and echovirus 11 are weaker than between the other two
viruses and their receptors. The faster dissociation rate of the echovirus 11·CD55
complex may be related to the finding that the interaction does not lead to structural
changes of the virus particle (114), as occurs with poliovirus and rhinovirus. The lower
dissociation rates for the poliovirus- and rhinovirus-receptor complexes may in part
reflect the time required for structural changes to occur. Elucidation of the high resolution
crystal structures of all three virus-receptor complexes should provide explanations for
the differences in kinetic parameters.
Why do poliovirus and rhinovirus have two classes of receptor-binding sites? One
possibility is suggested by a three-dimensional model of the poliovirus·sPvr complex
(Figure 15). In this model, domain 1 of sPvr contacts two major sites on the virus surface,
one in a cleft on the "south rim" of the canyon and a second on the side of the mesa on
the "north rim." Whether these two contact sites correspond to the two classes of binding
sites can be tested by carrying out kinetic and equilibrium binding studies on viruses with
amino acid changes in these areas (33). Since all contacts of Pvr with the virus involve
domain 1 (Figure 14), the finding of two classes of binding sites cannot be explained by
the involvement of Pvr domains 2 and 3. Two classes of binding sites might also be a
consequence of the structural flexibility exhibited by both viruses. Normally internal
parts of the poliovirus and rhinovirus capsid proteins have been shown to be transiently
- 82 -
poliovirus and rhinovirus with their cellular receptors leads to irreversible and more
extensive structural changes (12, 29, 65, 71). Antiviral drugs, such as WIN compounds,
which replace the lipid-like molecule in the hydrophobic pocket, are believed to block
uncoating of the capsid by rendering it structurally rigid. Binding of poliovirus to its
cellular receptor may cause release of the lipid-like molecule from the hydrophobic
pocket, allowing the capsid to undergo structural transitions necessary for binding and
entry. Such structural plasticity might explain the presence of two different classes of
binding sites on the virion. At lower temperatures, the higher affinity binding site is less
abundant compared with the lower affinity site. At higher temperatures, the relative
abundance of the higher affinity site is increased compared with the lower affinity site.
One explanation for these observations is that increased breathing of the virus at higher
temperatures results in the exposure of the higher affinity site. In addition, the interaction
between receptor and virus may induce a conformational change in the capsid that results
in exposure of the higher affinity binding site. In contrast to the findings with poliovirus
and rhinovirus, binding of echovirus 11 with CD55 can be described by a simple 1:1
binding model. Such behavior, which would be expected for the interaction of two
preformed binding sites, is consistent with the fact that the echovirus-CD55 interaction
does not result in detectable structural changes in the capsid (114).
Correlation of Cryo EM structure with mutational data
The interactions described by Cryo EM and image reconstruction are consistent
with mutational analyses (2, 16, 105) in the majority of mutations that affect receptor
- 83 -
loop, the C terminus of the D-strand, and the DE and FG loops of the receptor have been
associated with alterations in virus binding and the ability of the receptor to support
infections (2, 16, 105). However, five single-site mutations in the BC loop do not affect
binding, perhaps because none of these residues is essential (16). Similarly, several
mutations within the receptor footprint on the virus have been shown to alter the ability to
be neutralized by sPvr, bind wild-type receptor, or initiate infection with mutated
receptors (33, 35, 57, 88). These include mutations in the C-strand, EF loop, and C
terminus of VP1; in the N-terminal end of the smaller EF loop of VP2; and in the β-bend
(residues 58-60) and GH loop of VP3.
Not all of the capsid residues identified in the genetic studies cited above are
involved in contacts with Pvr. Those that are not-with one exception-are buried in
interfaces between subunits or on the inner surface of the capsid. The exception involves
mutations in the BC loop of VP1 that have been associated with mouse adaptation, ability
to use mutated receptor, and ability to establish persistent infection (reviewed in (117)).
The BC loop is well outside the footprint of the receptor in the road map (Fig. 14a), and
viruses in which it is replaced by a variety of heterologous sequences are viable.
Alterations in the BC loop are associated with significant changes in thermal stability and
the ability of the virus to undergo receptor-mediated conversions. It is therefore possible
that these mutations affect cell-entry steps after receptor binding (145).
A similar explanation has been proposed for mutations in residues that are buried
in interfaces, in the drug-binding pocket in VP1, or on the inner surface of the protein
shell (145). Indeed, most of these mutations have been shown to alter thermal stability or
- 84 -
several nonexposed residues (including residues 178 of VP3 and 177, 231, and 241 of
VP1) result in significant reductions in affinity for receptor, in competition assays with
wild-type virus (145). These data suggest that tight receptor binding may require minor
conformational changes in the virion. This suggestion is further supported by the recent
demonstration that the ability to bind receptor is ablated by capsid-binding drugs, such as
WIN51711, at low temperature but not at 37°C (41). These conformational changes may
be related to the "breathing" of the virus under physiological conditions (87). Since the
complexes studied here were formed by brief incubations at 4°C, they may represent the
initial state of receptor binding.
One possible consequence of the 60° turn between the long axes of d2 and d3 is
demonstrated in Figure 15e. If d3 were to emerge from the cell membrane with its long
axis normal to the plane of membrane, then d1 and d2 would be inclined at an angle of
30° relative to this plane. Given the binding aspect of Pvr to the virus (Figure 14c), this
configuration would orient a 5-fold axis perpendicular to the membrane, thus facilitating
multiple symmetry-related attachments of receptors around this axis. Moreover, it would
bring the mesa at the 5-fold axis close to the membrane. This juxtaposition is relevant to
the proposal that a continuous channel is formed along the 5-fold axis of the capsid and
through the membrane, as a mechanism for membrane attachment and RNA release (12).
An overview of the site of interaction
As forecast by chimeric receptor and point mutation analysis, domain 1 of sPvr
contains all contacts with the virus and approaches the capsid surface from the right as
- 85 -
pointing downward, toward the virus surface. Most of the interactions with the virus
involve the BC, C'C", DE, and FG loops (Figure 23). The BC loop inserts in a groove
defined by the smaller EF loop of VP2 (138-142), residues at the distal end of the GH
loop of VP1, and residues in the bulge at the C-terminal end of the GH loop of VP1 (234235). The DE loop contacts the residues at the N-terminal end of the GH loop of VP1
(214), the distal end of the GH loop of VP1 (224-226), and the GH loop of the 5-fold
related copy of VP3 (182-183). The FG loop contacts the C-strand of VP1, and the C'C"
loop of the receptor contacts the EF loop of VP1 (166-169). The latter contacts are the
only ones made with residues on the north wall of the canyon.
In addition to contacts involving loops at the N-terminal end of d1, the N-terminal
end of the D-strand and the C-terminal end of the E-strand (which are just below the d1d2 junction) contact the hairpin "knob" proximal to the B-strand of VP3 (58-60) from the
5-fold related protomer and residues near the C terminus of VP1 (293-297) from the
adjacent protomer (Figure 23). These resides are located in the "southeast" portion of the
receptor footprint (Figure 15a).
srr mutants possess varying degrees of resistance to sPvr
Many of the srr mutants predicted the site of interaction in the canyon of the
virus. To understand how the srr mutations confer resistance to neutralization by receptor
we determined the IC50 for each variant. The results indicate that there is a difference in
the level of resistance to neutralization by sPvr between the variants. The level of
resistance ranged from 0.0061 µM for wt. poliovirus to greater than 0.27 µM for the
- 86 -
observed phenotypes does not exist. The location of mutation on virus, accessibility of
mutation, reduction of 160S-to-135S upon binding to cell surface receptor, and a
decreased affinity of the mutant for cell surface do not reveal an obvious pattern in
relation to resistance to sPvr (Table 4, Figure 22). In order to understand how resistance
is conferred, the affinity of sPvr for the variants must be determined. Furthermore, it may
be necessary to determine the activation energies for the N to A conversion for the
variants in the presence and absence of receptor if the mutations modulate this
thermodynamic parameter instead of or in addition to decreasing the affinity for the
receptor.
Recombinant sPvr and soluble Pvr purified from human serum neutralize
poliovirus at similar concentrations
In the majority of studies presented in this work recombinant sPvr purified from
293 T cells was used. Limited quantities of naturally occurring soluble Pvr has been
purified from human serum, sCD155, and provided to us by Mark Denis laboratory.
Provision of sCD155 allowed us to compare the activities of recombinant and naturally
occurring receptor proteins. This study was done to insure that recombinant sPvr is not
acting in an artifactual manner in the biochemical and biophysical experiments. A sideby-side comparison of sPvr and natural sCD155 purified from human serum in a
neutralization assay demonstrated a remarkably similar profile and ID50 values. This
study indicated that recombinant sPvr is acting in a manner similar to the receptor
purified from human serum and it is suitable for biochemical and biophysical studies.
- 87 -
Kinetic analysis of the effect of poliovirus receptor on viral uncoating: the receptor
as a catalysis
In a 1996 review (145), James Hogle and colleagues hypothesized that the
poliovirus receptor may act as an enzyme using some of the energy of binding to lower
this free energy barrier of conversion and allowing conversion to occur at physiological
temperature. This theory is based on the observation that it is possible to reproduce the
transition from intact virions to the 135S particle by heating; this suggests that native
virion may represent a metastable intermediate in which the particle is trapped, by energy
barriers, from the path to lower energy. We have provided Simon Tsang and James Hogle
with sPvr. It is a necessary reagent to explore this hypothesis. Some of the results from
their experiments are presented in this section (143).
The Arrhenius equation was used as a mathematical tool to determine the energy
of activation (Ea) for the N to A transition. To employ the Arrhenius equation, the effect
of sPvr on the rate constant of the N to A transition of virus was examined by
incubations at various temperatures in the presence of 0.25 uM sPvr. Alteration from N to
A particle can be induced in vitro by warming the virus in the presence of millimolar
concentrations of calcium (38) without the formation of the empty capsid. The N to A
transition was monitored with an immunoprecipitation assay using a monoclonal
antibody (P1 McAb) raised against residues 24-40 of the capsid protein VP1. This
antibody does not recognize the native virion and has little or no affinity for the 80 S
particle (46, 142). In the experiments, the ratio of receptor to binding site at the
- 88 -
Figure 22.
Locations of srr mutations in the capsid structure. (a) An α-carbon
trace of a single poliovirus protomer viewed from the side. Sixty protomers form an
entire capsid. Note the peaks at the 5-fold and 3-fold axes of symmetry, and the canyon
that separates them. Surface srr mutations in VP1 are shown as yellow dots, green dots
indicate internal srr mutations, and residue numbers are listed. The light blue molecule
represents sphingosine bound in the hydrocarbon-binding pocket of VP1. (b) Two
protomers viewed from outside the virion, showing the interface between protomers.
Surface srr mutations in VP1 and VP3 are shown as yellow dots, white dots represent the
positions of two Sabin 3 ts-suppressing mutations, and residue numbers are shown.
- 89 -
Figure 23:
Diagram of domain 1 of Pvr (cyan), with β-strands labeled, abutting
inferred contact segments of the capsid proteins. Viral portions are shown as tubes,
with VP1, blue; VP2, yellow; and VP3, red. Residue numbers are provided as landmarks.
Black balls and colored numbers denote amino acids implicated by genetic analysis in
receptor binding. Similarly, Pvr residues shown by mutation to be important for virus
binding are listed (Left). The axes allow this view to be related to Figure 15d.
- 90 -
Relative
Resistance
Mutant
Name
ID50
µM)
(µ
Capsid
Protein
Residue
Number
Wt
Mutant
Structural
Location
Phenotype
Greatly reduced
binding to PVR;
normal 160S-to135S alteration
14
292
0.010
VP1
132
Met
Ile
Inaccessible in
lipid binding
pocket
5
211b
0.18
VP1
226
Asp
Gly
2
312
0.27
VP1
234
Leu
Pro
6
1072
0.17
VP1
236
Asp
Gly
11
332
0.053
VP1
241
Ala
Thr
4
1022
0.20
VP3
183
Ser
Gly
1
111c
>>
0.27
VP1
228
Leu
Phe
Accessible in G-H
loop
Accessible in G-H
loop, near
opening of lipid
binding pocket
Partially
accessible in G-H
loops, near the
opening of lipid
binding pocket
Inaccessible in H
strand, in fivefold
interface
Partially
accessible in
fivefold interface
Inaccessible, near
surface in G-H
loop
8
R252
0.069
VP2
215
Ser
Cys
7
151a
0.079
VP1
225
Gly
Asp
3
612
0.21
VP1
226
Asp
Asn
Accessible, in GH loop
9
382
0.063
VP1
231
Ala
Val
10
251a
0.056
VP1
241
Ala
Val
13
242
0.020
VP1
265
His
Arg
12
212
0.041
VP3
178
Gln
Leu
Partially
accessible, in G-H
loop
Inaccessible, in H
strand, in fivefold
interface
Internal surface,
near fivefold
interface,
contacting VP4
Inaccessible, in
fivefold interface
15
P1/M
0.0061
Inaccessible, in
fivefold interface
Inaccessible, near
surface in G-H
loop
Greatly reduced
binding to PVR;
160S-to-135S
transition is
reduced upon
binding to
receptor on cell
surface
Partial reduction
in binding to
PVR; 160S-to
135S transition
appears normal
Partial reduction
in binding to
PVR; 160Sto135S transition
is reduced upon
binding to
receptor
Surface accessibility determined by elevating the maximum radius of a sphere that contacts any atom within the residue
without contacting another atom in the structure. Residues were an accessible if they could be contacted by a sphere greater
than or equal to 3 Å in radius (Modified from review (31).
- 91 -
thermodynamics of poliovirus sPvr interactions are complex.
The available data suggest that the levels of receptor used in this study should be
sufficient to guarantee high occupancy of the available sites, but are probably insufficient
to guarantee full occupancy (60 sites/virion). Titration of varying concentrations of sPvr
demonstrated that further increases in the rate of conversion of virus could be achieved at
higher concentrations (data not shown). Limited amounts of sPvr were available at the
time of experimentation; therefore, the concentrations are less than sufficient to guarantee
full occupancy and maximal rates, and with minor caveats, as noted, the implications of
the results presented below are expected to be independent of changes in occupancy.
The Arrhenius equation states that for a first-order reaction obeying simple
transition states kinetics, the rate constant for a reaction is exponentially dependent on the
temperature: k=A exp (-Ea/RT), where k is the rate constant, Ea is the activation energy, R
is the gas constant (1.98 kcal/mol deg), and T is the temperature in Kelvin. The preexponential factor A is described by the relation: A = (kbT/h) exp (∆S†/R)
where Kb is Boltmann’s constant, h is Planck’s constant, and ∆S† is the entropy
difference between the ground state and the activated complex. Therefore, a plot of the
natural logarithm of the first-order rate constant versus –1/RT should yield a line with a
slope that is equivalent of Ea and a y-intercept that is proportional to ∆S.
The virus produces a linear Arrhenius plot in the presence of receptor; this
indicates that the first-order rate constants are dependent on a single exponential function
as required by simple transition state theory (Figure 21). Since the Arrhenius plots are
- 92 -
temperatures. Similar behavior has been previously reported for the N to A transition of
virus and virus –drug complexes in the absence of receptor (142). The addition of
receptor fragment decreases the Ea of the N to A transition by 50 kcal/mol, from 145
kcal/mol for virus alone versus 95 kcal/mol for virus +sPvr (Figure 21). Increasing the
number of receptor occupancy sites occupied on the virus might be expected to induce a
further decrease in Ea. As predicted, the receptor is behaving like a classic transition state
theory catalyst, accelerating the rate of the transition by lowering the activation barrier. A
kinetic model of the N to A transition can be seen in (143).
General rules for picornaviruses-receptor interactions
What basic principles can be deciphered from the information known about virusreceptor interactions? Picornaviruses must undergo two processes to infect a host cell:
first, they must bind to the cell; second, they must uncoat the genomic RNA and release
in into the host cell cytoplasm. Picornaviruses have evolved to use two different types of
receptors with different consequences on the virus uncoating and entry processes. For this
discussion, receptors that only bind virions will be called “binding receptors”. Receptors
that can both bind virus and cause the release of the virus genome will be called
“uncoating receptors”.
Viruses that use uncoating receptors such as HRV3, poliovirus, and CAV21 differ
in four aspects when compared to picornaviruses that use binding receptors. One, HRV3,
poliovirus, and CAV21 have receptor occupancy sites that reside within the canyon (13,
58, 111, 149). Two, the canyons of HRV3 and poliovirus are flexible and each has two
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The two affinities are likely to reflect the plastic nature of the canyon region. Three, the
soluble receptor fragments are sufficient to promote conversion of the virus (4, 29, 51,
97, 99). Four, membrane bound forms of the uncoating receptors for HRV3, poliovirus
and CAV21, not only concentrate virus at the cell surface, but also promote conversion
and allow entry. Binding receptors for picornaviruses contrast significantly with primary
receptors. One, binding receptors do not bind the canyon. It has been suggested that the
echovirus 11 receptor, DAF, binds at the 2-fold axes of symmetry of the capsid (S.M.
Lea, personal communication). The human rhinovirus serotype 2 (HRV2) binds its
receptor, VLDL-R, on the small star shaped dome on the icosahedral 5-fold axis (60).
Two, the receptor occupancy site on the virus is preformed and stable with a single
affinity for its receptor. For example, echovirus 11 has only one affinity for its receptor as
determine by surface plasmon resonance using a soluble CD55 fragment (84). Third,
soluble binding receptors do not promote conversion of the virus particle; for example,
echovirus 7 is not converted to an altered particle by binding to its binding receptor, DAF
(114). Fourth, membrane bound binding receptors act only to sequester virus on the cell
surface but are not sufficient to mediate entry (15).
These observations, taken together, show a clear pattern emerging. Receptor
type, binding or uncoating, is governed by a distinct set of parameters: location of
receptor binding on the virus surface; receptor uncoating capacity; and receptor entry
mediating ability. Most importantly, uncoating receptors bind the canyon and, perhaps
because of the plastic properties of this region on the protein shell, this interaction
initiates uncoating. This plasticity may permit the canyon to “open-up”, “Pinch”, or
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Figure 24: Arrhenius plots for the N to A transition in the presence and absence of
sPvr. Averaged values of natural log of the rates of conversion ln k from (143) were
plotted as a function of –1/RT, the slope of each line is the energy of activation for
conversion. Bold lines and dashed lines correspond to data collected in the presence of
sPvr and absence of sPvr, respectively (From (143)).
- 95 -
These are likely to be the changes in the protein shell necessary to initialize the release of
VP4 and RNA.
A possible exception to these rules is O1 FMDV that binds hepran sulfate (HS) in
the pit in the capsid surface. The pit has a similar location to the canyon on the virus
surface but is shallower. HS binding to the pit does not cause conversion as would be
expected by these rules. However, this finding is likely to be an exception because the
depression is too shallow or because the ligand is a sugar moiety.
Model of poliovirus interaction with cell surface
How does poliovirus use the energy of interaction for entry? On the virus-side of
the receptor, where the receptor contacts the canyon, the surface area of contact is large
and specific. This seems to allow a decrease in the activation energy and catalysis of
conversion of the metastable virus. On the cell surface-side of the receptor, poliovirus
has been observed by electron microscopy in invaginations on the membrane (151).
However, entry of poliovirus is not dependent on clathrin-mediated endocytosis (40).
Therefore, we propose a simple model (termed: bind, remodel, release) for virus
entry that allows for more efficient genome release into the host cells cytoplasm (Figure
25). The model is predicated on three observations: one, multiple receptors can bind virus
simultaneously; two, poliovirus is found in invaginations on the cell surface; three, the
free energy of interaction for a single receptor interacting with a virus is –10 Kcal/mol.
Furthermore, the total energy of virus interaction with the cell surface is likely to be
much higher because of multivalency. The energy generated by the multivalent
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membrane. This would allow for membrane dimple formation similar to what has been
postulated for influenza interaction with the cell surface (138). Since the virus can release
its genome in the absence of membrane, its direction of release is likely to be orientation
non-specific. Therefore, the gathering of the membrane around the virus particle should
allow for a more efficient genome release and infection.
Conclusions and future directions
The result presented in this thesis, poliovirus receptor recognition: visualization;
kinetics; and thermodynamics, are initial answers to questions formulated 16 years ago
upon determination of the crystal structures of poliovirus and rhinovirus. In his review (7)
of these crystal structures, David Baltimore discussed seven major questions that arose by
the structural renderings. Two of these questions are the birthplace of this thesis: First,
what imparts receptor specificity to the virion? The similarity of polio- and rhinovirus
argues that the receptor specificity is “encoded” in the details of surface architecture, not
in gross features. Second, how are particles “disassembled”? The receptor must somehow
find a key site in the virion to affect this conversion reaction. Answers to these questions
began after the poliovirus receptor was identified. Use of the receptor in conjunction with
the infectious poliovirus cDNA clone gave researchers the opportunity to correlate
mutational analysis with detailed structural knowledge; in one key study, solubilized
receptors were used to isolate viral variants that were resistant to neutralization. This
provided the initial insights into how the capsid interacts with receptors.
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biochemical, and structural studies. Verifying genetic data using cryoelectron
microscopy, we have used soluble Pvr to demonstrate that the canyon is the viral surface
feature that “encodes” the receptor docking site for poliovirus. Interestingly, major group
HRVs and CAV21 also use the canyon to contact their cellular receptors. The reason
there are conserved canyon docking sites for some picornaviruses and not for others, such
as minor group HRVs is unclear, but it is likely to be related to the mechanism of
uncoating.
We have shown that soluble receptor purified from mammalian cells is sufficient
to “disassemble” poliovirus. The details of the uncoating mechanism are unknown.
Determination of the crystal structure of sPvr and poliovirus-sPvr complex will be
important steps in delineation of the mechanism. These studies will allow the interaction
to be understood at the atomic level, which is important for understanding the mechanism
of any biochemical reaction. Clearly the canyon is the key site that the receptor must
contact to “disassemble” virus and the pocket factor, which is located beneath the canyon
floor, plays a role in this process. How the pocket is dislodged from the virus to allow
uncoating is unclear. Pocket factor may be squeezed out of the canyon by receptor
induced structural capsid changes. Or, the receptor may contact the pocket factor directly
and this may initiate its release.
Once the atomic details of the interaction are understood it may be possible to design
mutant sPvr forms that are able to “trap” the virus in intermediate stages of uncoating.
For example, if in the crystal structure three distinct regions of the receptor are contacted
by the virus then mutations of specific amino acids at each of these regions separately
- 98 -
uncoating. An experimental procedure may be designed which can be used to “trap” the
virions in interesting states such as partial RNA release, partial VP4 release, or viral
expansion. These states may be visualized by Cryo EM and transitions the virus goes
through may be better understood.
Cryo EM image reconstruction of the conversion product catalyzed by sPvr will
be useful in determining the mechanism of uncoating. Although the heat conversion
products, 80S and 135S, have been visualized. They may possess structural differences
when compared to the products catalyzed by sPvr. SPR has demonstrated that the relative
abundance of the two binding sites varies with temperature and is, therefore, dynamic. It
is likely the two binding sites for the receptor on the poliovirus type 1 capsid are the first
clues to a mechanism of uncoating. The two binding sites may correspond to docking
sites for domain 1 of the receptor on the viral capsid, as predicted by a model of the
poliovirus-receptor complex. Alternatively, the binding sites may be a consequence of
structural breathing, or could result from receptor-induced conformational changes in the
virus.
Evidence that the conversion process is the mechanism of virus genome release
into the cell is compelling. Further evidence that this process is the mechanism of entry
would be to demonstrate that sPvr, not tethered to the cell surface, is able to mediate
entry. Recent studies have shown that a soluble form of the subgroup A avian sarcoma
and leucosis virus (ASLV-A) receptor (sTva) is able to mediate entry of ASLV-A
without being tethered to the cell surface (39). This result suggests the previously
described receptor induced conformational changes on ASLV-A are likely to be involved
- 99 -
Figure 25:
Model for poliovirus entry: Bind, remodel, and release.
- 100 -
yield a similar result providing more evidence that conversion by Pvr is the mechanism of
RNA introduction into the host cell.
The questions of how poliovirus enters the cell are likely to remain for many
years. Biophysical and biochemical technologies, such as, cryoEM, SPR, isothermal
titration calorimetry, and crystallography, as well as cell biological techniques which
visualize dynamic cellular processes, are advancing rapidly and will greatly assist the
animal virologist in answering entry related questions. It is my hope the eradication
effort will not cease poliovirus research before the inevitably dazzling answers to these
questions are revealed.
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