Journal of General Virology (1991), 72, 1021-1029.
1021
Printed in Great Britain
Changes in macromolecular synthesis of gypsy moth cell line
IPLB-Ld652Y induced by Autographa californica nuclear polyhedrosis
virus infection
David Guzo, 1. Edward M. Dougherty, 1 Dwight E. Lynn, 1 Susan K. Braun 1
and Ronald M. Weiner 2
1United States Department of Agriculture, Agricultural Research Service, Insect Biocontrol Laboratory, Building O11A,
Room 214, BARC-West, Beltsville, Maryland 20705 and 'University of Maryland, Department of Microbiology,
College Park, Maryland 20742, U.S.A.
The aberrant replication of the Autographa californica
multiple-enveloped nuclear polyhedrosis virus
(AcMNPV) in the Lymantria dispar cell line IPLBLd652Y was used as a model system for the investigation of factors regulating baculovirus host specificity. A
previous study of this system indicates that viral gene
expression in infected cells is extremely attenuated and
subsequently all cellular and viral protein synthesis is
inhibited. In the present study, infection of IPLBLd652Y cells with A c M N P V photochemically inactivated in situ resulted in a rapid reduction in cell mitotic
indices and cell growth, as well as inducing a series of
distinct morphological changes in these cells. At the
molecular level, infection with inactivated virus, followed by pulse labelling with [3H]thymidine, resulted
in a rapid [0 to 2 h post-infection (p.i.)] and permanent
inhibition of host cellular DNA synthesis. Assays of
cellular DNA polymerases in isolated IPLB-Ld652Y
nuclei confirmed the reduction in cellular DNA
synthesis observed in intact cells and indicated an
initial (0 to 2 h p.i.) reduction in the activity of
aphidicolin-sensitive DNA polymerases. Activity of all
cellular DNA polymerases was inhibited at later times
p.i. Host cell protein synthesis was completely inhibited after 48 h p.i. Treatment of inactivated virus
and virus-infected cells with various chemical and
physical factors (i.e. pH and temperature) or lysosomotropic agents revealed that virus entry into cells and
fusion of endocytic vesicles (containing virus) with
lysosomes were essential for suppression of cellular
macromolecular synthesis. The possible involvement
of structural components of the A c M N P V virion in
these effects is discussed.
Introduction
californica multiple-enveloped nuclear polyhedrosis virus
The nuclear polyhedrosis viruses (NPVs) have been
shown to infect a variety of economically important
lepidopteran insect species (Ignoffo, 1968; Gr6ner,
1986; Dougherty, 1987); however, many baculoviruses
effectively replicate in and kill only one or a limited
number of host species. Although some of the barriers to
normal baculovirus replication in mammalian cell lines
and cells of certain non-target insect species are defined
(Carbonell et al., 1985; Carbonell & Miller, 1987), the
factors which restrict baculovirus replication within the
lepidoptera are poorly understood.
In our laboratory, we have been interested in
identifying the molecular barriers which determine the
host range of baculoviruses including the Autographa
(AcMNPV). An important aspect of this research has
involved characterizing the abnormal or aborted
AcMNPV replication in a cell line (IPLB-Ld652Y)
derived from the gypsy moth, Lymantria dispar, which is
not normally infected by the virus (McClintock, 1986a).
This cell line supports a limited degree of viral gene
expression, followed by a total shutoff of host cellular and
viral protein synthesis at approximately 20 h postinfection (p.i.). We report here that infection with
AcMNPV, the DNA of which had been inactivated in
situ, resulted in a permanent and irreversible inhibition
of synthesis of macromolecules in IPLB-Ld652Y cells. In
this cell line, therefore, normal viral replication may be
inhibited by a viral component(s) which exerts an
inhibitory effect on cell macromolecular synthesis. The
0000-9630 © 1991 SGM
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1022
D. G u z o a n d o t h e r s
relevance of these data with regard to the factors
regulating AcMNPV replication in the gypsy moth is
discussed.
Methods
Cell lines and virus. The ovarian, pupa-derived L. dispar cell line
IPLB-Ld652Y (Goodwin et al., 1977) was maintained in IPL-52B
medium (Hazelton Research Products) as previously described
(Goodwin & Adams, 1980). IPLB-LdEIta cells (Lynn et al., 1988) were
maintained in BML-TC/10 medium (Gardiner & Stockdale, 1975).
Unless otherwise indicated, cells were incubated at 28 °C.
All infection experiments used a specific plaque-purified AcMNPV
clone, 6R (McClintock, 1986b). Virus titres were determined by a
modification of the endpoint dilution assay (Brown & Faulkner, 1975)
in TN-368 cells (Hink, 1970).
Virus infections. Twelve-well plates (Linbro, Flow Laboratories) were
inoculated with 2.5 × 105 log phase IPLB-Ld652Y cells per well. After
attachment, cells were inoculated with normal or inactivated
AcMNPV (see Results; Inactivation o f the A c M N P V genome) or nonoccluded virus (NOV) at m.o.i.s ranging from 0.5 to 100 TCID~o per
cell. Unless otherwise indicated, cell cultures were infected at an m.o.i.
of 50. After a 1 h infection period, the virus inoculum was removed, the
cells were rinsed three times with fresh tissue culture medium, and were
incubated in the appropriate medium for the duration of the
experiment.
For generation of AcMNPV from IPLB-LdEIta cells, log phase cells
were infected at an m.o.i, of 10. NOV was subsequently harvested 4
days p.i. and stored at - 2 0 °C until use.
Inactivation of the viral genome. Short-wave u.v. or photochemical
inactivation of viral genomes in situ, followed by infection of host cells
with the inactivated virus, has been used previously to distinguish
between cellular effects induced by virion components and those by
viral gene products (Fenwick & Walker, 1978; Nishioka & Silverstein,
1978). Two different methods were utilized to inactivate the AcMNPV
genome. The psoralen derivative, trioxsalen (4,5,8-trimethylpsoralen;
Sigma) was used in combination with long-wave u.v. light (360 nm) to
crosslink double-stranded nucleic acids in situ as previously described
(Guzo & Stoltz, 1985). AcMNPV nucleic acids were also inactivated by
short-wave (254 rim) u.v. irradiation (Griego et aL, 1985). Inactivation
of the viral genome was confirmed by endpoint dilution assays on TN368 cells.
Additionally, slot blots of total RNAs extracted from both
psoralen/u.v.- or short-wave u.v.-inactivated virus-infected cells were
probed with nick-translated, whole viral DNA or AcMNPV genespecific probes (see below) to determine the level, if any, of virusspecific transcription.
Cell growth and mitotic indices. Cell growth rates were measured by
initially plating 6-0 x 10a IPLB-Ld652Y cells per well into 12-well
plates. Cells were counted in a Neubauer haemocytometer with each
data point representing the average of five separate counts. Mitotic
cells were identified directly by phase microscopy or after staining with
the vital stain hydroethidine (Polysciences) following the manufacturer's instructions.
Tritiated thymidine incorporation. The effects of treatments on rates of
cellular DNA synthesis were determined by pulse labelling with
[3H]thymidine. Typical labelling experiments used IPL-52B or TNMFH medium (Hink, 1970) with 5~Ci/ml of [3H]thymidine (NEN).
After labelling, cells were lysed in 2% sodium sarcosinate and the
nucleic acids precipitated with 5 ~ trichloroacetic acid. The labelled
DNA was then immobilized on Whatman glass fibre filters and the
amount of incorporated [3H]thymidine was determined with a Searle
Mark Itl Scintillation counter (Searle Analytical). Labelling experiments were repeated three times.
DNA synthesis in isolated nuclei. Characteristics of cellular DNA
synthesis in isolated nuclei from uninfected, AcMNPV-infected and
inactivated virus-infected IPLB-Ld652Y cells were examined. Nuclei
from infected and uninfected cells were isolated by a previously
described method (Wood et al., 1982). Conditions for optimum DNA
synthesis in intact nuclei in vitro were as described by Flore et al. (1987).
Activities of different DNA polymerases were determined using
chemical inhibitors with demonstrated antagonistic effects against
specific insect DNA polymerases (Miller, 1981; Sakaguchi & Boyd,
1985; Mikhailov et al., 1986; Wernette & Kaguni, 1986.). Aphidicolin,
an inhibitor of ~ and 6 DNA polymerases (Kornberg, t980), ddATP
(inhibits primarily p and ~ polymerases) and N-ethylmaleimide
(inhibits ~ and ~ polymerases) were all obtained from Sigma.
Isotopic labelling of A c M N P V NOV. [3H]Thymidine-labelled
AcMNPV NOV was produced in TN-368 cells. Three days p.i., the
NOV was collected and purified as described previously (McClintock,
1986b). An aliquot of the virus sample (1/10th) was counted in a Searle
Mark III Scintillation counter to determine the amount of incorporated
radioactivity.
IPLB-Ld652Y cells were infected for 1 h with 3H-labelled AcMNPV
or 3H-labelled inactivated virus at an m.o.i, of 10. After infection, cells
were rinsed three times with sterile Locke's saline and refed with
complete medium. Two h after infection cells were briefly treated with
Pronase (10 min at 27 °C; Calbiochem) to remove any external virus,
after which nucleic acids from whole cells or cell nuclei were
precipitated with 5 ~ trichloroacetic acid and immobilized on
Whatman glass fibre filters. The amount of internalized virus was
determined by dividing the c.p.m, of 3H in whole infected cells by the
total c.p.m, of 317I in the original virus inoculum.
Infections in the presence o f drugs. The effects of inhibitors and
lysosomotropic agents on inactivated virus infection were determined
by preincubation of the cells for 30 min with each drug followed by
inactivated virus inoculation as described above. The virus inoculum
and fresh medium controls also contained each drug at equivalent
concentrations. All inhibitors and lysosomotropic agents were obtained
from Sigma. Leupeptin, an inhibitor of certain acidic lysosomal
proteases, was used at 1 mM; vinblastine, a microtubule poison which
can inhibit fusion between phagocytic vacuoles and cellular lysosomes,
was used at 0.1 ram. Propylamine and NH4C12, lysosomotropic weak
bases, were used at concentrations of 25 mM and 20 mM, respectively.
The effects of drug treatments on cellular DNA synthesis in
inactivated virus-infected IPLB-Ld652Y cells were determined by
incubating infected and uninfected cells for 2 h p.i. in the presence of
5 ~tCi/ml [3H]thymidine. [3H]Thymidine incorporation into cellular
DNA was determined as described above. Incorporation experiments
in the presence of inhibitors were replicated three times. Potential
virucidal effects associated with the lysosomotropic agents or inhibitors
were assayed in TN-368 cells using AcMNPV exposed to each
particular drug for 1 h before it was collected by centrifuging at
t00000g (2 h) and resuspended in fresh TNM-FH medium. In some
experiments, inactivated virus was pretreated with polyclonal
AcMNPV-specific or monoclonal anti-64K protein AcMNPV-specific
antibodies (provided by Dr Loy Volkman, University of California,
Berkeley, Ca., U.S.A). Inactivation of normal AcMNPV by antibody
treatment was confirmed by endpoint dilution assays on TN-368 cells.
Isolation of nucleic acids. AcMNPV DNA was extracted from
purified occlusion bodies as previously described (McClintock et al.,
1986a). AcMNPV-speeific ptasmids containing the IE-1 (Guarino &
Summers, 1986) or IE-N (Carson et al., 1988) genes were generously
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Baculovirus virion component
provided by Dr Linda Guarino, Texas A & M University, College
Station, Tx., U.S.A. Total RNA fractions from uninfected, AcMNPVinfected and inactivated virus-infected IPLB-Ld652Y cells were
extracted by the hot phenol-guanidinium hydrochloride method
(Maniatis et al., 1982). Any contaminating DNA was removed by
digestion for 1 h at 37 °C with RNase-free DNase (20 ~tg/ml; Fluka).
Preparation of RNA slot blots. Cellular RNA samples were denatured
in 50% formamide and 10 x SSC for 15 min at 60 °C. Samples were
immediately cooled on ice and applied to nitrocellulose filters in a
Minifold II Slot blotter (Schleicherand Schuell). RNA filters were then
air-dried (1 h) and baked at 80 °C for 2 h in a vacuum oven before
hybridization.
Nick translation and hybridization conditions. Whole viral DNA or
plasmids were labelled with [ct-3zp]CTP (NEN) by nick translation
(Rigby et al., 1977) to specific activities of > 1.0 × 108 c.p.m./pg.
Immediately prior to hybridization, DNA probes were denatured by
boiling (I0 min) in 3 volumes of hybridization buffer (see below) and
cooled on ice.
Nitrocellulose(or Nytran) filters were processed and hybridized with
32p-labelled viral DNA by previously described methods (Stoltz et al.,
1986). Filters were air-dried and analysed by autoradiography.
Radiolabelling of cell proteins and SDS-PAGE. Log phase IPLBLd652Y cells were added to Linbro 12-Multiwelltrays at a concentration of 2-5 x 105 cells/well and were inoculated with inactivated NOV
at an m.o.i, of 50 TCID50 for 1 h. After infection, cells were rinsed
twice and refed amino acid-free IPL-52B medium containing 40 pCi
[35S]methionine (NEN) per ml. Ceils were pulse-labelled for 4 h
periods p.i. Radiolabelled cells were pelleted, suspended in disruption
buffer (McClintock et al., 1986a) and boiled for 5 min. Cell samples
were stored at -20 °C until use. Proteins were separated by SDSPAGE (Laemmli, 1970). Staining and autoradiography techniques
were as described previously (McClintock et al., 1986a).
Results
Inactivation o f the A c M N P V
genome
I n a c t i v a t i o n of the A c M N P V g e n o m e by p h o t o c h e m i c a l
m e a n s was initially confirmed by e n d p o i n t dilution
assays using T N - 3 6 8 cells. T r e a t i n g A c M N P V with
trioxsalen plus long-wave u.v. or with short-wave u.v.
light was equally effective in i n a c t i v a t i n g virus, each
causing a 105-fold r e d u c t i o n i n titre. T h e absence of
A c M N P V - s p e c i f i c gene activity was confirmed by
p r o b i n g slot blots of total R N A s extracted from
uninfected, A c M N P V - i n f e c t e d a n d i n a c t i v a t e d virusinfected I P L B - L d 6 5 2 Y cells with 32p-labelled whole
A c M N P V D N A a n d A c M N P V gene-specific probes.
Typical results are presented in Fig. 1. N o detectable
A c M N P V - s p e c i f i c gene activity was observed at a n y
time p.i. with trioxsalen/u.v, or short-wave u.v.-inactivated virus. I n all s u b s e q u e n t e x p e r i m e n t s , results
o b t a i n e d using trioxsalen/u.v.- a n d short-wave u.v.i n a c t i v a t e d virus were identical a n d both types of
i n a c t i v a t e d virus p r e p a r a t i o n s will be referred to as i-v.
Actual entry of i-v into host cells was confirmed by
infecting IPLB-Ld652Y
cells w i t h
3H-labelled
(a)
1
2
1023
3
0
6
S
12
24
48
72
(b)
0
2
6
12
24
Fig. 1. Slot blot of total RNAs from uninfected (lane 1), AcMNPVinfected (lane 2) and trioxsalen/u.v.-inactivated (lane 3) virus-infected
IPLB-Ld652Y cells. Approximately 25 pg of total RNA was spotted
per sample at each time (h) period shown on the left. (a) The blot was
probed with 32p-labelledAcMNPV DNA and exposed to X-ray film
for 24 h. (b) The blot was probed with the 32p-labelled IE-I gene
sequence and was exposed to X-ray film for 72 h.
A c M N P V . O f the label p r e s e n t in the infectious
A c M N P V inocula, 1 5 . 1 % + 2 . 3 % (±S.D.) was internalized in host cells, a n d 9-1% + 3.6% of the isotope
p r e s e n t in the i n a c t i v a t e d virus inocula was internalized.
T h e a m o u n t of isotope i n t e r n a l i z e d in cells as a result of
virus i n a c t i v a t i o n was not statistically different (Stud e n t ' s t-test, P ~< 0-05).
Changes in cell morphology and growth rates
Cell morphology progressively c h a n g e d after infection of
I P L B - L d 6 5 2 Y cells with i-v (Fig. 2). By 18 h p.i. some
cellular c l u m p i n g a n d b l e b b i n g was observed; f o r m a t i o n
of large dense cellular aggregates had occurred by
36 h p.i. T h e m a j o r i t y of cell aggregates s u b s e q u e n t l y
(48 h p.i.) detached from the flask surface.
I n f e c t i o n with i-v also caused a p e r m a n e n t suppression
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1024
D. Guzo and others
I
I
i
I
(a)
20
i
x
I
I
i
i
i
i
(b)
~'6
2
i
i
.(c)
8O
60
40
20
0
Fig. 2. Changes in IPLB-Ld652Y cell morphology induced by i-v
infection. (a) Uninfected IPLB-Ld652Ycells. (b) IPLB-Ld652Y cells
18 h p.i. with i-v, (c) IPLB-Ld652Y ceils 36 h p.i. with i-v. Note the
formation of dense cellular aggregates. Bar marker represents 100 ~tm.
of cell growth and a reduction in mitotic indices (Fig. 3 a
and b). Despite the rapid suppression of cell growth,
extensive cell mortality was not observed until 96 h p.i.
(Fig. 3c). Most r~on-viable cells also retained their
structural integrity until 7 to 10 days p.i.
Protein synthesis inhibition
Changes in protein synthesis in IPLB-Ld652Y cells
infected with inactivated virus were monitored by pulselabelling cellular proteins with [3SS]methionine (Fig. 4).
Polypeptide profiles in uninfected and inactivated virusinfected IPLB-Ld652Y cells were alike until after 24 h
p.i. when most cellular protein synthesis was inhibited in
i-v-infected cells. At no time were A c M N P V infectionspecific proteins observed in i-v-infected IPLB-Ld652Y
cells.
0
i
I
24
48
I
72
Time (h)
I
96
120
Fig. 3. (a) Changes in IPLB-Ld652Y cell growth rates, (b) mitotic
indices and (c) viability, induced by infection with AcMNPV (Z2x),
trioxsaten/u.v.- ([]) and short-wave u.v.- (0) inactivated AcMNPV.
Untreated control cells are represented as (O). Error bars represent
standard deviations for each data point.
To determine whether the lack of protein synthesis in
i-v-infected cells was a consequence of changes in the
ability of infected cells to internalize the radiolabelled
methionine, uninfected and i-v-infected cells (at 72 and
9 6 h p.i.) were pulse-labelled with [35S]methionine
(40 ~tCi/ml) for 4 h, rinsed thoroughly and the amount of
internalized radioisotope was counted. N o significant
difference in the amounts of internalized radioisotope
between uninfected and i-v-infected cells was noted
(data not shown).
Inhibition of DNA synthesis
Infection with i-v permanently inhibited I P L B - L d 6 5 2 Y
D N A synthesis in cell cultures (Fig. 5). Cellular D N A
synthesis was immediately affected by infection, with
incorporation of [3H]thymidine reduced to 51.3~+_
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Baculovirus virion component
1
2
3
4
~
5
~
6
7
8
~-~
9
10
1025
11
÷~ ' ?
:: i?. :ill i
Fig. 4. Autoradiogram of electrophoreticallyseparated [3SS]methionine-labelledproteins from i-v-infected IPLB-Ld652Y cells.
Infectedcellswerelabelledfor consecutive4 h periodsfrom 1 to 4 (lane2), 4 to 8 (lane 3), 8 to 12 (lane4), 12to 16(lane 5), 16to 20 (lane6)
and 20 to 24 (lane7) h p.i., and subsequentlyfor 4 h periodsat 44 to 48 (lane 8), 68 to 72 (lane 9) and 92 to 96 (lane 10)h p.i. Lanes 1 and 11
show protein profiles for uninfected IPLB-Ld652Yceils.
6 . 7 ~ of control levels by 0 to 2 h p.i. (Fig. 5b). Levels of
incorporation of [3H]thymidine in infected cell cultures
were reduced to approximately a third of normal by 12 h
and to permanently below 1 0 ~ of normal levels by 24 h
p.i. (Fig. 5a).
In order to determine whether specific cellular D N A
polymerases were selectively inhibited by i-v infection,
cellular D N A synthesis in isolated IPLB-Ld652Y nuclei
was assayed in the presence of various D N A polymerase
inhibitors. Data presented in Table 1 indicate that the
product synthesized was insensitive to RNase A but was
sensitive to DNase I. Also, synthesis of D N A was
inhibited by heat treatment of nuclei or by the presence
of sodium pyrophosphate. D N A synthesis in uninfected
cells was predominantly the product of aphidicolinsensitive cellular D N A polymerases (i.e. c( and/or 6
polymerases). Incubation of nuclei with d d A T P and Nethylmaleimide indicated that the remaining D N A
synthetic activity was primarily due to cellular/3 and 7
D N A polymerases. The proportions of D N A synthetic
activity attributed to the different types of D N A
polymerases in normal uninfected IPLB-Ld652Y cells
also agree closely with those ~previously observed in a
variety of eukaryotic cells (Kornberg, 1980). At 2 h p.i.
with i-v, cellular D N A synthesis was primarily aphidicolin-insensitive, thereby indicating that the majority of
the D N A synthesized was the product of cellular fl and 7
D N A polymerases. D N A synthetic activity at 12 and
24 h p.i. also appeared to be primarily the product of
cellular fl and 7 type polymerases. Levels of D N A
synthesis in nuclei from AcMNPV-infected cells at 2 h
p.i. were similar quantitatively and qualitatively to those
in nuclei from i-v-infected cells. Further assays of D N A
synthetic activity in normal AcMNPV-infected cells at
12 and 24 h p.i. were not done owing to the presence of
virus-encoded D N A polymerase activity at these times
p.i.
In order to study whether the i-v infection induced
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D. Guzo and others
1026
Table 1. Effects of various treatments on DNA polymerase activity* in nuclei from uninfected, AcMNPV-infected or short-wave
u.v.-inaetivated A c M N P V (u.v./AcMNPV)-infected IPLB-Ld652Y cells
AcMNPV
u.v./AcMNPV
Inhibitory treatment
Uninfected
2 h p.i.
2 h p.i.
12 h p.i.
24 h p.i.
Control (no treatment)
Heat (100 °C/5 rain)
50 ~tg/ml DNase I
50 l.tg/ml RNase A
2 % (w/v) sodium pyrophosphate
50 ~tM-ddATP
10 ~tM-N-ethylmaleimide
50 p.g/ml aphidicolin
100% (341 790 c.p.m.)*
5%
4%
94 %
3%
77 %
32 %
18 %
100% (101 622)
100% (88950)
-
100% (130450)
23 %
45 %
68 %
100% (39960)
16%
30
66 %
24 %
29 %
79 %
19%
26 %
87 %
* Activity in all cases is expressed as the percentage of c.p.m, incorporated in nuclei from uninfected or virus-infected cells not subjected to any
inhibitory treatment.
Table 2. Effects of chemical inhibitors and lysosomotropic
15 t- (a)
Ps Ac
i
AcMNPV
x
10
~.
weak bases on i-v infection-induced cellular DNA synthesis
inhibition
[ 1 ~ u.v. Ac
1
Control
5I
b.-
0 ~
0-12
12-24
24 48
48-72
72 96
96 120
Treatment
[3H]Thymidine incorporation (%)
Control (no treatment)
Inactivated AcMNPV (i-v)t
i-v/vinblastine
i-v/leupeptin
i-v/NH4CL.
i-v/propylamine
100 (11650)*
59 + 3.1
89 + 3.6
104 + 4.0
61 +_1"5
57 + 2-5
* Activity is expressed as the percentage of c.p.m. (average _+
standard deviation) in uninfected cells over a 2 h incorporation period.
Virus was inactivated by short-wave u.v. irradiation as described
in Methods.
A c M N P V replication. Virus from I P L B - L d E I t a cells
was i n a c t i v a t e d p h o t o c h e m i c a l l y a n d used to infect
I P L B - L d 6 5 2 Y cells. C h a n g e s in cell growth, m o r p h o l o g y
a n d m a c r o m o l e c u l a r synthesis were identical to those
i n d u c e d with A c M N P V g r o w n in T N - 3 6 8 cells.
15
~<--,
i
c:)
x
1o
Effects of various treatments on the ability of i-v to alter
IPLB-Ld652 Y cell biology
b..-
0-2
2-4
4 6
6-8
8-10
10-12
Fig. 5. Changes in [3H]thymidine incorporation within 120 h p.i. (a)
and within the initial 12 h p.i. (b) in IPLB-Ld652Y cells as a result of
infection by AcMNPV, trioxsalen/u.v.-inactivated (Ps Ac) and shortwave u.v.-inactivated AcMNPV (u.v. Ac). Error bars represent
standard deviations for each data point.
effects o n D N A a n d p r o t e i n synthesis, as well as on host
cell growth a n d morphology, were some f u n c t i o n of the
A c M N P V g r o w n in T N - 3 6 8 cells, A c M N P V N O V was
grown in a second cell line ( I P L B - L d E I t a ) permissive for
T o characterize the events involved in the i-v infectioni n d u c e d suppression of host cellular m a c r o m o l e c u l a r
synthesis, virus inocula were treated with various
physical or chemical agents prior to b e i n g used for
infection.
T r e a t m e n t of i-v with heat (56 °C, 20 min), acid p H
(pH 3.0, 1 h), chloroform or n e u t r a l i z i n g a n t i - 6 4 K
protein m o n o c l o n a l a n t i b o d y e l i m i n a t e d the infectioni n d u c e d effects on D N A a n d p r o t e i n synthesis, cell
morphology a n d cell growth. I n c u b a t i o n o f i-v with n o n n e u t r a l i z i n g a n t i - 6 4 K p r o t e i n m o n o c l o n a l antibodies,
however, had n o effect o n the capacity of i-v to induce
changes in I P L B - L d 6 5 2 Y cell biology. Overall, a n y
t r e a t m e n t that i n h i b i t e d virus e n t r y into cells also
e l i m i n a t e d the effects o n cellular D N A a n d p r o t e i n
synthesis, cell m o r p h o l o g y a n d cell growth rates.
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Baculovirus virion component
Medium from which i-v was removed by centrifugation
(150000g for 3 h) had no discernible effect on IPLBLd652Y cells. The rapidity of DNA synthesis inhibition
implied that an early event in the virus infection
pathway, possibly involving the uptake and entry of
virus particles into host cells, may interfere with host
cellular DNA synthesis. Inhibitors that blocked fusion of
lysosomes with phagocytic vesicles (vinblastine) or
inhibited acidic proteases (leupeptin) abolished the
ability of i-v to block cellular DNA synthesis (Table 2).
Alternatively, i-v-infected cells incubated with lysosomotropic weak bases which prevent fusion between viral
envelopes and the membranes of cellular phagolysosomes by raising the intralysosomal pH (NH4CI2 and
propylamine) exhibited rates of DNA synthesis not
significantly different from those in i-v-infected cells in
the absence of inhibitors. AcMNPV preincubated with
any of the drugs used here was capable of initiating
normal infections in TN-368 cells (data not shown).
Discussion
The barriers that limit the host range of baculoviruses
within the lepidoptera can conceivably involve any
aspect of viral attachment and entry, viral uncoating,
DNA replication or expression of the viral genome.
Although the barriers to normal replication in any
particular baculovirus/non-permissive host combination
are likely to be complex, a more thorough understanding
of these factors may eventually permit the generation, by
recombinant means, of baculoviruses with broadened
host ranges.
In the present study we have expanded upon our
previous investigation of the barriers to AcMNPV
replication in IPLB-Ld652Y cells (McClintock et al.,
1986a). In that study, AcMNPV virions were shown to
be able to enter host cells, uncoat and initiate normal
DNA replication. Expression of the viral genome is
however extremely attenuated. Numerous cellular c.p.e, s
are noted in infected cells; these include a cessation of
cell growth, cell clumping and an abrupt and total
inhibition of cellular protein synthesis by 20 h p.i. We
demonstrate here that all of the c.p.e.s previously
associated with AcMNPV infection in IPLB-Ld652Y
cells could be induced by inactivated virions. The
possibility that these effects are artefacts resulting from
some membrane incompatibility between AcMNPV
grown in a cell line from Trichoplusia ni (TN-368) and
gypsy moth cells is considered unlikely as inactivated
virus grown in IPLB-LdEITa cells exhibits similar
activities in IPLB-Ld652Y cells. Also, similar activities
associated with AcMNPV virions in another gypsy moth
cell line (derived from the larval fat body tissue) have
1027
recently been demonstrated (E. M. Dougherty, unpublished observations).
The most rapid effect of inoculation with inactivated
virus was a decline in cellular DNA synthesis, similar to
that observed during normal baculovirus replication
(Knudson & Tinsley, 1978) in which 12~ of total DNA
synthesis was cellular at 8 to 12 h p.i. This decline in
DNA synthesis was evidenced by several lines of data,
including a rapid reduction in cellular incorporation of
pH]thymidine in whole cells, a reduction in DNA
synthesis by aphidicolin-sensitive DNA polymerase(s) in
nuclei in vitro, and by a suppression of cell growth and
mitosis. We therefore consider the DNA synthesis
inhibition to be a significant effect induced by infection
of IPLB-Ld652Y cells with inactivated AcMNPV.
Indeed, we have been able to duplicate these effects
artificially only through addition of high concentrations
of potent DNA synthesis inhibitors (i.e. 20 to 40 ~tg/ml
aphidicolin) to the tissue culture media.
The rapid reduction in cell DNA synthesis implied
that some early step(s) in virus infection (i.e. adsorption,
penetration or uncoating) was involved in inducing this
effect. Previous research on early events in baculovirus
infection indicates that the primary route of virus entry
into cells is probably by adsorptive endocytosis and that
subsequent uncoating of virions can be inhibited by
lysosomotropic agents which interfere with low pHdependent fusion of virus envelopes and phagolysosomal
membranes (Volkman & Goldsmith, 1985). The elimination of the i-v infection-induced DNA synthesis inhibition effect by an inhibitor of fusion between lysosomes
and phagocytic vesicles (vinblastine) and by an inhibitor
of lysosomal acidic proteases (leupeptin), indicates that
the fusion of cellular lysosomes with phagocytic vesicles
(containing virus) is probably a critical step in the
generation of the factor(s) which suppresses DNA
synthesis. Also, since lysosomotropic weak bases did not
affect i-v inhibition of DNA synthesis, the release of
nucleocapsids into the cytoplasm may not be necessary
for the cellular DNA synthesis inhibition. Possibly an
AcMNPV virion component(s) was released or enzymically modified by the action of phagolysosomal proteases
on the virion nucleocapsid in the absence of normal viral
uncoating. The mechanism(s) by which DNA synthesis
is inhibited in i-v-infected cells is not completely
understood, although it can be hypothesized that an
antagonistic interaction between inactivated virions or
virion components and cellular DNA polymerases may
occur. Alternatively, the cellular DNA synthesis inhibition may be a secondary effect of some unidentified
infection-induced toxic effect or some incompatibility
between host cells and AcMNPV virions.
The most prominent feature of AcMNPV infection of
IPLB-Ld652Y cells involves the total inhibition of
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1028
D. Guzo and others
cellular and viral protein synthesis by 20 h p.i. (McClintock et al., 1986a). The current study suggests that some
AcMNPV virion component(s) inhibits cellular protein
synthesis in IPLB-Ld652Y cells. The longer time
required for the total inhibition of protein synthesis in
inactivated virus-infected cells (48 h compared to 20 h
with normal AcMNPV) also implies that some
AcMNPV-specific gene product modulates early protein
synthesis inhibition. This is consistent with results found
in herpes simplex virus infection of vertebrate cell lines
in which cellular macromolecular synthesis inhibition is
apparently regulated by both virion-associated factors
and viral gene products (Sydiskis & Roizman, 1966;
Honess & Roizman, 1973; Nishioka & Silverstein, 1978;
Kwong et al., 1988). The types of interactions between
AcMNPV virion components and cellular transcription/
translation pathways are not known. Conceivably, some
component(s) of the inactivated AcMNPV virion might
interfere directly or indirectly with cellular protein
synthesis by degrading host mRNAs or by binding to
essential cellular factors involved in normal transcription/.translation. Alternatively, the presence of inactivated virions in the cell may induce some cumulative
injury to the host cell which could result in protein
synthesis inhibition. Additionally, AcMNPV-specific
protein synthesis in IPLB-Ld652Y cells has been
demonstrated to be attenuated prior to total protein
synthesis inhibition (McClintock et al., 1986a) and it is
possible that virion components may also interfere with
early viral protein synthesis in this cell line.
The mechanism(s) by which baculoviruses inhibit host
cell macromolecular synthesis is not well understood. In
this study, we have demonstrated that inactivated
AcMNPV virions, in the absence of detectable viral gene
expression, could induce a total shutoff of host cell
protein synthesis and a significant reduction in cellular
DNA synthesis. The data, while not conclusive, also
suggest that a virion component(s) may mediate these
effects. Obviously, the identification and characterization of any baculovirus virion factor(s) which inhibits
host cell macromolecular synthesis could be a significant
step in the development of compounds specifically
designed to inhibit DNA and protein synthesis in insect
cells.
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