Intracellular events following the infection of different cell types with vesicular stomatitis subviral particles

VIROLOGY

135,266-27X (1984)

Intracellular Events following the Infection of Different Cell Types with Vesicular Stomatitis Subviral Particles CLAIRE MARTINET-EDELIST,**‘” CHRISTINE TUFFEREAU,?’

VICTOR DEUTSCH,*,l NICOLE GENTYS

AND

*In&itut de Mkmin&gk, BZt 409; ~Labo&oire de G&&ique, Bit. loo; and $Labomtoire de Biochimie physique, B& .bsS,Univemiti Par&&WE, 91.@5&say Ced.ex, France Received January 5, 1984 accepted February 28, 1984 Vesicular stomatitis virus (VW) subviral particles (nucleocapsids and G-depleted particles) were used to infect various cells (chicken embryo, HeLa, and BHR21 cells). These particles bind to and penetrate into host cells; the association of G-depleted particles to cells was even better than that of normal virions. The parental genomes of subviral particles and virions were degraded at the same rate in the infected cells. Nevertheless, these subviral particles had a very low infectivity, synthesized very little viral macromolecules, and had very little, if any, effect on the various host cells used. Furthermore, subviral particles could be rescued in chicken embryo cells by uv-irradiated VSV virions, demonstrating that subviral particles actually penetrated into cells, and that their arrested cycle could be unblocked up to a certain point. On the other hand, subviral particles were not rescued in HeLa cells, suggesting a dependence on the host cell system.

through coated vesicles to the endosomes, where the acidic pH triggers the fusion of The virion of vesicular stomatitis virus the viral envelope with the endosome (VSV) consists of a nucleocapsid and a lipid membrane, leading to transfer of the nuenvelope with two associated, virally coded cleocapsid into the cytoplasm and infection proteins, the glycoprotein (G) and the ma(Marsh et al, 1983). The envelope proteins trix protein (M) (Wagner, 1975). The three are generally thought to be essential for other viral proteins and the genome RNA virus entry and budding (Heine and constitute the nucleocapsid; the nucleoSchnaitman, 1971). In addition, G and M protein (N), in association with the genome proteins have been shown to exert a control RNA, functions as template, whereas the on viral RNA synthesis; this regulation has large (L) and NS proteins are the subunits of the RNA-dependent RNA polymerase been demonstrated in viva, using temperature-sensitive mutants (Martinet et c& (Emerson and Yu, 1975). 1979), and in vitro (Combard and PrintzCellular phosphatidylserine appears to A&, 1979). The mechanism of this control be important for VSV viral attachment to is not yet clear. On the other hand, the VERO cells (Schlegel et aL, 1983). Morepresence of G and M proteins is not an over, when VSV infects a host cell, it seems absolute requirement since VSV nucleoto follow the same endocytic pathway as capsids are infectious, although only with other enveloped viruses (Matlin et d, 1982); a very low efficiency (Cartwright et al, 1969, the transcribable nucleocapsid is then un1970a, b; Deutsch, 1970). Preliminary excoated and released into the cytoplasm. As periments have shown that nucleocapsids demonstrated with Semliki Forest virus (Helenius et aL, 1980), after binding to re- could bind to and penetrate into cells like virions, and G-depleted particles did it even ceptors on the cell surface it is internalized better (Genty et a& 1980). We therefore undertook a detailed study of the pseu1 Present address: Laboratoire de GQnetique des docycle induced by VSV subviral particles; Virus, CNRS, 91199 Gif/Yvette, France. 2To whom reprint requests should be addressed. binding, penetration, production of partiINTRODUCTION

0042~6622/84 $3.00 Copyright Ail rights

8 1%4 hy Academic Press, Inc. of reproduction in any form reserved.

266

SUBVIRAL

PARTICLES

OF VSV

267

(i) Inocula were diluted in saline buffer containing bovine serum albumin (BSA) (500 ~1 of each dilution was added to 2 * lo6 cells) and allowed to bind at 4” or at 37’, under gentle agitation, for indicated times. Cells were then washed three times with MATER1AJ.S AND METHODS cold saline buffer, scraped off, and pelleted Cells. HeLa cells and primary cultures at 1000 g for 5 min. The pellet-associated of chicken embryo cells (CEC) have been radioactivity was measured after pellet described previously (Printz-An6 et al. dissociation in 200 ~1 of Soluene and ad1972). BHK21 cells clone 21 was isolated dition of scintillation fluid (Picofluor) in by A. Flamand by successive dilutions of an Intertechnique SL40 spectrometer. an uncloned BHK21 cell population. (ii) The vH$rridine inocula were added Virions and s&&al particles. A heat- to cells and allowed to bind to the cells resistant strain of VSV (Indiana serotype) (BHK21 and HeLa cells) at room temperdescribed previously (Combard et al. 197’7) ature for 45 min under mild agitation. was used throughout this study. Subviral Minimum essential medium (MEM) was particles, i.e., nucleocapsids and G-depleted then added; 1 hr later (i.e., 1 hr p.i.), the particles, were extracted from purified vi- cells were washed twice to remove desorbed rions using Triton X-100, either in 500 mM particles. Then, cytoplasmic extracts in Tris buffer, 250 mil4 KCl, 5 m&f magnesium RSB (reticulocyte saline buffer: 10 mM acetate, 0.1% mercaptoethanol, or in 10 mJ4 NaCl, 10 mlM Tris, 1.5 m&f MgCI2, pH 7.4) Tris according to Combard and Printz-A& were prepared by addition of 1% sodium (1979). G-depleted particles were also pre- dodecyl sulfate (SDS) as described previpared by octylglucoside extraction accord- ously (Printz-An& et a& 1972). The raing to the procedure of Newcomb and dioactivities of the trichloroacetic acid Brown (1981). The yields were titrated on (TCA)-precipitable material of the inocula CEC as described by Flamand (1970). and of the cytoplasmic extracts were deUltraviolet (WV)irradiation, VSV prep- termined after filtration on 0.45-pm nitroarations or cells were exposed to uv irra- cellulose filter (Millipore Corp.). diation (253.7 mm) as described previously, under conditions which produced a dose Protein electrophwresis. Proteins were rate of 50 erg mm-’ set-’ (Deutsch, 1975). precipitated overnight at 4’ by the addition Electron microscope. Virions and subviral of 2 vol of ethanol to cell extracts after particles were routinely observed, after centrifugation at 10,000 rpm for 30 min; the pellets were dissolved in a solution of negative staining with phosphotungstic acid (pH 7), with a Philips EM 300 micro- 62.5 mM Tris-HCl, pH 6.8, 1 M urea, 1% SDS, 1% mercaptoethanol. Samples were scope at 80 kV. Preparation of [5Tfjwidine-lubeled par- boiled for 2 min and subjected to electroticles. CEC were infected with VSV at a phoresis for 4 hr at 30 mA on discontinmultiplicity of infection (m.0.i.) of 0.1 uous, Tris-buffered SDS-slab gels consisting of 10% polyacrylamide resolving gel plaque-forming unit (PFU) per cell. After 45 min of adsorption at 30”, Eagle’s me- and 3% stacking gel according to Laemmli’s dium with 2 &i/ml of C3H]uridine was procedure (1970), modified by Banerjee et added and cells were incubated at 37”. The al (1974). The gels were then fixed in VSV virions harvested after 24 hr of in- methanol/acetic acid/water (4/l/15, vol/ cubation were purified, and subviral par- vol/vol). Gels were stained with Coomassie ticles were prepared as already described blue; the protein bands present in virions and subviral particle preparations were above. Biding to cc& Two different techniques scanned with a DD2 Kipp and Zonen denwere used, both with rH]uridine-labeled sitometer at 500 to 550 nm. The gels used for analyzing intracellular proteins, laVSV particles (virions, G-depleted partibeled with r5S]methionine, were dried and cles, or nucleocapsids).

cles, fate of the parental genome, viral macromolecular synthesis, effect of these particles on cell metabolism, and rescue by uv-irradiated virions.

268

MARTINET-EDELIST ET AL.

finally exposed to Fuji X-ray medical film at -70”. Analysis of wird RNA !q sucrose grdti fractionation. Cytoplasmic extracts prepared as described above were fractionated by centrifugation in a 15 to 30% sucroseSDS gradient at 21,000 rpm for 15 hr at 1’7” in a Spinco SW27-1 rotor as described elsewhere (Combard et al. 1974). Analysis of viral RNA by hybridization The cells were infected with [3H]uridinelabeled virions or subviral particles. At different times after infection in cold medium, cell extracts were prepared and the synthesized, unlabeled RNA was annealed with labeled virion RNA as described by Saghi and Flamand (1979). Uridine penetration. At different times following infection of CEC by VSV virions or subviral particles, the culture medium was replaced by fresh medium containing [3H]uridine at a concentration of 5 * lop7 1M. The dish cultures maintained at 37” were gently agitated during the 15-min incubation period. The monolayers were then washed four times with cold saline buffer and treated for 30 min at 4” with 5% TCA. The cells were scraped off the dish and centrifuged. The radioactivity associated with TCA-precipitable material (pellets were dissolved in Soluene) and the radioactivity present in TCA-soluble extracts was determined. Antiserum Antispike Indiana serotype antiserum was prepared as previously described (Deutsch, 1976).VSV particles were treated with antiserum at room temperature for 30 min at a final dilution of 11 40. The reaction was stopped with 5% calf serum. After adsorption, the infected cells were washed three times; MEM was then added and cells were incubated at 37”. Chemicals. Minimum essential medium was purchased from Eurobio, and F15 medium from Flobio. Actinomycin D was a generous gift from Merck, Sharp & Dohme. [3H]Uridine (sp act, 20 Ci/mmol) was obtained from CEA, Saclay, France, and L[35S]methionine (sp act, 1100 Ci/mmole) from Amersham, U. K. Octylglucoside (noctyl-p-D-glucopyranoside) and Triton X100were products from Sigma. Soluene and Picofluor were purchased from Packard.

RESULTS

Ana1~si.s of the Subviral Particles VSV viral nucleocapsid-like particles could be seen with the electron microscope in suspensions of nucleocapsids and G-depleted particles prepared with Triton X100. As to the G-depleted particles prepared with octylglucoside, they yielded compact structures somewhat similar in appearance to intact virions, but without spikes (data not shown), confirming the results of Newcomb and Brown (1981). The protein composition of subviral particles and virions was determined on slab gels, and each sample was scanned (Fig. 1). In the nucleocapsid preparation the G protein was undetectable, whereas around 2% of the M protein was still present as compared to virions. A residual peak of G protein corresponding roughly to 5% of the normal amount was observed on both profiles obtained with the G-depleted particles, while only a slight decrease of the M protein peak could be noticed. Furthermore, lipid analysis showed that less than 10% of the total virion lipids was present both in nucleocapsids and in Gdepleted particles, no matter which detergent was used (R. Salesse, personal communication). Binding of VSV Subwiral Particles to Cells Figure 2 indicates the kinetics of the binding of nucleocapsids, G-depleted particles, or virions to monolayers of CEC at 4 or 37”. The radioactivity associated to cells was higher at 37 than at 4”, probably as a consequence of particle internalization. Furthermore, it can be noticed that the percentage of radioactivity bound to cells was significantly higher-about twice-in the case of G-depleted particles than in the case of virions or nucleocapsids. Internalization was checked by treatment of infected cells with proteinase K (0.5 mg/ ml) at 4“ during 45 min postinfection (not shown); it is known that this treatment removes virions which are associated to the cell surface (Matlin et al, 1982). Following 1 hr infection by virions at 37”, 75% of the radioactivity was proteinase K re-

SUBVIRAL

PARTICLES A

Virions

N

269

OF VSV N

NucleOcapSids

M

I

I I

L NS

JdJd S depleted

M

\

c G depleted

particles

(octvlglvcoside) _ -.

i

t

1

(Triton

particles x 100)

i

M

L J&-L_1

G

NS II

migration

-

FIG. 1. Protein analysis of VSV subviral particles. Nucleocapsids were prepared with 0.5% Triton X-100 in 50 r&f Tris buffer, 250 m&f KCl, 5 n-&f magnesium acetate, 0.1% mercaptoethanol, pH 7.6. G-depleted particles were obtained by treatment with 0.5% Triton X-100 or with 66 mM octylglucoside, both in 0.01 M Tris buffer, pH 7.6. Aliquots (20 81) were loaded onto each well of a 10% slab gel and electrophoresed for 4 hr at 30 mA. After staining with Coomassie blue, each sample was scanned with a densitometer.

sistant whereas, after infection at 4”, only 15% of the radioactivity bound to cells was proteinase K resistant. On the other hand, proteinase K treatment could detach neither nucleocapsids nor G-depleted particles from cells whatever the temperature of absorption (37 or 4’), possibly suggesting a different process of binding of these particles to cells. The binding of subviral particles to cells was also studied after desorption, and the figures obtained were submitted to statistical tests. Virions and subviral particles labeled with rmridine were used to infect BHK21 (clone 21) and HeLa cells. CEC were not used in these experiments because of their high ribonuclease content. Binding

was expressed as the percentage of the TCA-precipitable radioactivity of each cytoplasmic extract versus that of the corresponding inoculum (Table 1). Binding of the G-depleted particles to HeLa and BHK21 cells was much higher than the binding of virions or nucleocapsids. This confirms the results described above using CEC. A distribution-free statistical test (rank test of Mann-Whitney (Jacobson, 1963)) showed that the observed differences were significant with a risk of l%, for all experiments taken together. On the contrary, the same statistical test showed that the figures obtained with nucleocapsids and virions were not significantly different, even with a risk of 5%.

MARTINET-EDELIST

270

ET AL.

NC

30

60 time

60

30 post

infection

30

60

(minutes)

FIG. 2. Rate of association of virions and subviral particles to CEC. culture medium; [aHjuridine-labeled virions, nucleocapsids, or G-depleted cpm/plate; m.o.i. 10 PFU/cell for virions) were then allowed to bind at agitation for indicated times. Cell-associated radioactivity is expressed inoculum radioactivity.

CEC were washed with particles (500 pl; &O,OOO 4O (0) or 37” (0) under as the percentage of the

roughly to the same number of PFU as in the subviral particle inoculum (m.o.i. N 10d3 PFWcell). The curves obtained with subviral particles were relatively close to the curves Viral production induced by subviral particles was studied in different cell types; obtained with virions at a very low m.o.i., CEC, BHK21, and HeLa (Fig. 3). Purified except that for the first hours after infection the curves of subviral particles seemed virions were employed at two different concentrations as controls; (i) virions from to be lower. The same results were found the undiluted stock that was used to pre- whether subviral particles were treated or pare the subviral particles (m.o.i. = 200 not with antispike (= antiglycoprotein) PFU/cell), and (ii) virions with a very low VW antiserum. On the contrary, the control curves obtained with the virions at an multiplicity of infection, corresponding Kinetics of Production of Infectious Particles by Cells Ir4fected with Subviral or Intact Particles

TABLE

1

BINDINGOFSUBVIRALPARTICLESTOCELLS BHK21

HeLa Type of particles

Exp. 1

Exp. 2

Exp. 3

Exp. 4

Exp. 1

Exp. 2

Exp. 3

Exp. 4

Exp. 5

Virions G-depleted particles Nucleocapsids

6.5 7.3 2.2

1.5 13 4.6

2.2 9 0.6

6 13.6 3.5

4.3 10.3 3

4.5 8.8 2.1

1.6 7.3 1.7

3.6 19.6 6

1.4 5.6 0.7

Note. [‘H]Uridine-labeled subviral particles and virions (1 cpm = 5.10’ genomes) were used to infect HeLa and BHK21 cells; 1 hr p.i. cells were washed twice to remove desorbed particles. Then, cytoplasmic extracts in RSB with 1% SDS were prepared and precipitated with 5% TCA. Binding of the particles to cells is expressed as the percentage of radioactivity of the cytoplasmic extracts versus the corresponding input.

SUBVIRAL

1

10

20

PARTICLES

10

1

T I ME

(hours

271

OF VSV

20

1

10

20

p.i.)

FIG. 3. Time course of the production of infectious virus by several cell types infected with VSV subviral particles or virions at 37’. The m.o.i. for virions was 200 or l-10-* PFWcell; the m.o.i. for subviral particles was equivalent to 300 PFWcell. Particles were allowed to adsorb for 45 min at room temperature. Then, cells were incubated at 37“. At 1 hr p.i., cells were washed twice and the medium was changed to remove desorbed particles. Aliquots (0.2 ml) were taken at intervals and titrated on CEC. Symbols are (the titration of each inoculum is plotted by an arrow near its symbol) l , virions; A, very diluted viral suspension; 0, nucleocapsids; A, G-depleted particles.

m.o.i. of 206 PFU per cell were much higher says rescue of subviral particles only was than all other curves. Nevertheless at 16 carried out after these particles had been hr p.i. all final virus yields were of the same treated with antiserum, as indicated in Table 3. The titrations were carried out at order of magnitude. It can be concluded that, following bind- 6 hr p.i. to avoid superinfections by virions ing to cells, nucleocapsids and G-depleted produced by the cells infected with subviral particles had a very low infectivity that particles (Fig. 3). In BHK21 cells the 6-hr was not due to the residual activity of con- yields were very low; the data were stataminating virions. This infectivity was not tistically analyzed and discarded because figures were too variable. The results obdestroyed by antiglycoprotein antiserum for subviral particles, whereas the infec- tained in CEC and HeLa cells are plotted tivity of very diluted viral suspensions dis- in Table 3. On HeLa cells the rescue indices appeared completely (Table 2). Therefore, were low, whatever the particles used the intracellular events induced by these (subviral particles or virions at a very low m.o.i.); using the distribution-free statisparticles had to be studied further. tical test of Mann-Whitney, the figures were found not to differ statistically from Rescue of Subviral Particles by VV-Irracontrols, even with a risk of 5%. On the diated Viricms contrary, on CEC, the rescue indices were The penetration of subviral particles into higher with subviral particles than with cells and their possible reuse were checked virions at a very low m.o.i.; the figures obby means of rescue experiments. Since an- tained with the virions at a very low m.o.i. tiserum-treated virions did not give any are significantly different from those obinfectious particles (Table 2), in most as- tained with the subviral particles (with a

272

MARTINET-EDELIST TABLE ACTION OF ANTISPIKE

ET AL. 2

ANTISERUM ON VSV PARTICLES

Very diluted viral suspensions

G-depleted particles

Nucleocapsids

Exp. 1

Exp. 2

Exp. 3

Exp. 1

Exp. 2

Exp. 1

Exp. 2

Inoeulum without AS with AS

2150 ~5”

280 <5’

12,500 <5a

350 180

55 35

369 320

2850 350

Yields without AS with AS

-

-

27,506 <5”

650 75

115 500

50 165

1000 235

Note. Infected CEC were incubated at 37’; yields harvested at 6 hr p.i. or the corresponding titrated as described under Materials and Methods. The results were expressed in PFU/ml. ’ 15, i.e., no plaque was counted without dilution.

inoculum

were

risk of 1% using the distribution-free sta- were prepared and analyzed on 15-30s sutistical test of Mann-Whitney). Therefore, crose-SDS gradients (Fig. 4). Cytoplasmic it seems that at 37” subviral particles were extracts from uninfected cells, mixed with rescued in CEC by uv-irradiated virions, genome-labeled nucleocapsids, were anawhereas they were not in .HeLa cells. This lyzed as a control; the 40 S RNA peak (geindicates that subviral particles did pen- *nome size) was predominant. RNA profiles etrate into the cells. of infected HeLa cell extracts were like this control, whereas profiles of BHK21 cell extracts exhibited a second peak, correFate of the Parental Genmne sponding roughly to a velocity of 28 S. On Genome-labeled subviral particles and the other hand, the profiles obtained with virions were used to infect BHK21- (clone cells infected with subviral particles were 21) and HeLa cells; CEC could not be used very similar to those obtained .with wildowing to their .high ribonuclease content. type virions; the profiles at 5 hr p-i. were At 1 and 5 hr p.i., cytoplasmic extracts lower than at 1 hr pi., indicating that degTABLE

3

RESCUE OF SUBVIRAL PARTICLES BY UV-IRRADIATED WILD-TYPE VIRIONS Type of the infected cells

Very diluted viral suspensions

G-depleted

particles

Nucleocapsids

CEC

0.2-4.9-1.520.79-0.040.34

40-71*-14*-41*-52*2.4*-3.7+-52*

5.3-20*-10*-14*-10*-2.6*16.4*-9*-2.7*

HeLa

1.52-2-2.15

l-7-1.28*-0.27*-0.27*

0.3-0.22-2.4*-0.73*-0.55*

Note Mixed and single infections were carried out under the same conditions as in Fig. 3. UV dose was 8000 erg mme2. * G-depleted particles and nucleocapsids were treated before use with VSV antispike antiserum at a final dilution of l/40. Ihe supernatants of the cells were harvested at 6 hr p.i. and titrated on CEC. The results are given in terms of complementation index, C, i.e., the ratio of the mixed infection yields to the sum of the single infection yields. Each experiment was repeated several times and all the results are given in the table.

SUBVIRAL

I

BHK

(clone

PARTICLES

21) Cells

I

273

OF VSV

HeLa

cells

I

FIG. 4. Fate of the parental genome of VSV subviral particles and virions in BHK21 (clone 21) and HeLa cells. PHJUridine-labeled particles were used at the following m.o.i.: 400 PFWcell for virions and equivalent for G-depleted particles, or multiplicity equivalent to 100 PFWcell for nucleocapsids. In order for all inocula to roughly contain the same number of particles, their concentrations were adjusted by measuring their radioactivity. Infection was performed under the same conditions as in Fig. 3, at 3’7O. Cytoplasmic extracts were prepared at 1 and 5 hr p.i., and analyzed on a sucrose gradient as under Materials and Methods. Arrows in the figure indicate the peak positions of the cellular ribosomal18 S and 28 S markers. As a control, cytoplasmic extracts from uninfected cells were mixed with genome-labeled nucleocapsids, A. The other symbo!s are 1 hr p.i., 0; 5 hr p.i., 0.

radation of RNA genomes took place. But this degradation from 1 to 5 hr p.i. was analogous in all cases and not specific, whatever the infecting particles. Once more, significantly higher radioactivity of G-depleted particles at 1 hr p.i. was found, clearly showing a better association of these particles to cells. Polarity of RNA Synthesized in the Cells I$ected by S&viral Particles It was shown before that it was possible to determine the percentage of labeled viral genome undergoing transcription and/or replication by measuring the increase of

resistance to digestion with ribonuclease A (Saghi and Flamand, 19’79).We took advantage of this observation to measure the ability of G-depleted particles to transcribe and/or replicate. Monolayers of BHK21 (clone 21) cells were infected with genomelabeled, G-depleted particles and virions. The nucleic acids of the infected cells were extracted at 0, 2, 4, and 6 hr p.i. The percentage of [3H]ribonuclease resistance of the RNA was determined before any annealing (Fig. 5a). The parental genome RNA extracted from cells infected with Gdepleted particles exhibited very little, if any, resistance to digestion by ribonuclease A, whereas virions exhibited a normal,

MARTINET-EDELIST a

before

the infected cells was performed, followed by digestion with ribonuclease A. The quantity of (+) strands that were present in the cytoplasm was calculated from the number of counts per minute after hybridization. The results after annealing were expressed in “genome equivalent” mass copies per infected cell (Fig. 5b). For the virion-infected cells, the curve increased and reached a plateau corresponding roughly to 2 - lo* genome equivalents per cell; markedly different, the curve observed with the G-depleted particle-infected cells was very low, increasing slightly only at 6 hr p.i., from 102to 4 * 102 genomes equivalent per cell.

annealing

z

3 b

,A

after

ET AL.

annealing

/-

Protein @pathesis

J 1

1

I

2

4

6

T I ME

p.i. (Hours)

FIG. 5. Production of viral-complementary RNA in cellsinfected by VSV G-depleted particles and virions. Total unlabeled RNAs were extracted at intervals from cells infected with G-depleted particles or with virions (m.o.i. = 100 PFU/cell or equivalent). As the infecting particles were labeled with [dH]uridine in their genomes, the ribonuclease resistance of the RNAs was determined without annealing (a). The production of VSV-complementary RNA was measured by annealing the unlabeled RNAs extracted from infected cells to an excess of ‘H-labeled RNA extracted from purified VSV virions, as explained in the text. Specific activity of the genome RNA was 1 cpm = 3 - 106 molecules (b). Symbols: 0, virions; A, G-depleted particles.

increase of ribonuclease resisto 30%. This indicates that most intracellular genomes from G-depleted particles did not undergo transcription and/or replication. The determination of the actual level of synthesis of molecules complementary to viral genomes was then carried out. An excessof labeled genomes of known specific activity was added to aliquots of the cell extracts. Hybridization of these labeled genomes with total RNA extracted from significant tance, up

Slab gel electrophoresis was used to study protein synthesis in BHK21-infected cells. One can see in Fig. 6 that cellular protein synthesis was not inhibited by subviral particles or by control virions employed at very low m.o.i. (lOA PFU per cell). Presence of all viral proteins and good inhibition of cellular proteins could be seen on electrophoregrams of extracts of cells infected with virions at a m,o.i. of 100 PFU per cell. We then tried to lower the cellular background in order to see whether or not cells infected with subviral particles synthesized viral proteins, be it in small amounts. To this end, cells were uv irradiated with 2000 erg mmp2. Although very little cell protein synthesis was observed in control, uninfected cells, no viral protein synthesis was detectable even at 6 hr p.i. in cells infected with subviral particles. Viral proteins were normally manufactured in virion-infected cells (at high m.o.i.); they could be separated by electrophoresis and revealed by autoradiography (data not shown). Efects of Subwird Particle I?$ection on Host Cells (i) Morphology of the cells. It did not change during at least 6 hr after infection by subviral particles, whatever the cells used (CEC, HeLa, or BHK21 cells) (not shown). On the contrary, VSV virions rap-

SUBVIRAL

PARTICLES

275

OF VSV DISCUSSION

Our experiments (Fig. 2 and Table 1) demonstrated that VSV nucleocapsids bound to cells similarly to virions, whereas G-depleted particles did so at a much higher level, whatever the cells used (CEC, HeLa or BHK21 cells). This result was rather surprising, since it is generally considered that the spikes constituted by G protein (Cartwright et al, 19’70a, b) were necessary to attach virions to host cells (Cartwright et al. 1969; Schloemer and Wagner 1974,1975). Similarly, an increase in receptor-binding activity was found by Manjunath and SaYram (1982) after partial deglycosylation of the human polypeptide hormone, choriogonadotropin. Furthermore, binding of VSV subviral particles and normal virions was more efficient at FIG. 6. Intracellularprotein synthesis after infection 37” than at 4”, suggesting an internalizawith virions or subviral particles. Cells were infected tion at 37” as already described for the either with virions (m.o.i. N 100 PFU/cell), with subvirions (Matlin et al. 1982). But, after bindviral particles (G-depleted particles or nucleocapsids ing at 4”, whereas around 80% of the bound at a m.o.i. equivalent to that of the virions), or with virions were removed by proteinase K, very dilute viral suspensions (m.o.i. s lOmaPFWcell). subviral particles were not. An attachment Labeling lasted for 30 min with 20 &i/ml of [%l]methionine. Aliquots (20 ~1) of cell lysates were of subviral particles to phosphatidylserine loaded onto each well and electrophoresed for 4 hr could explain this last result; but, in that at 30 mA. Positions of the five viral proteins are incase, attachment to phosphatidylserine dicated by arrows. Abbreviations are C, intracellular would not be mediated by the VSV G protein synthesis in uninfected cells, control; V, viprotein. The apparent discrepancy with rions; VDV, very dilute viral suspension; NC, nucleothe trypsinization experiment of Schlegel capsids; G, G-depleted particles. (Schlegel et al, 1983) could be explained by the use of different cell types and different idly changed morphology of the cells experimental techniques. Our result might indicate a partially different process of (Genty and Bussereau, 1980). (ii) Eflect of VSV s&viral particles and binding to the cells for subviral particles virbzs 012uridine uptake by chick embryo and virions; similarly to subviral particles, cells. It has been shown that infection of choriogonadotropin hormone is not reCEC by VSV virions rapidly inhibits the moved from its receptors by proteinase K incorporation of uridine into RNA by in- (N. Genty, unpublished observations). On terfering with uridine uptake (Genty, 1975; the contrary, infectivity of both subviral Genty and Dragun, 1983). Inhibition of particles was very low as compared to viuridine uptake does not require virus rep- rions (Fig. 3), but was not due to the few virions remaining in these preparations lication and may be induced by parental virion (Genty, 1975). In CEC, neither nu- since treatment of subviral particles with cleocapsids nor G-depleted particles in- anti-G antibody did not prevent productive hibited uridine uptake and cellular RNA yields (Table 2). This has been demonsynthesis until 5 hr p.i. (not shown). This strated previously for the VSV nucleocapsids by Cartwright et al. (1969, 1970a, b) type of experiment has not been attempted and Deutsch (1970). Likewise, this obseron HeLa or BHK21 cells, as their infection by VSV virions does not alter uridine up- vation does not contradict the results obtained by Schnitzer et al. (1979), who have take (Genty, 1975).

276

MARTINET-EDELIST

found that the G-depleted particles yielded by cells infected with the mutant ts045(V) were not infectious. In order to explain these results, one could first suppose that subviral particles were degraded in infected cells at a faster rate than normal virions. This hypothesis was rejected because Fig. 4 shows that the parental genome RNA of virions and subviral particles were, in fact, degraded at the same rate. Therefore, the parental G protein (and perhaps some other part of the viral envelope) seems to have a crucial role at the beginning of the viral cycle. This very low virion production seemed, in fact, the consequence of weak viral macromolecular synthesis, very little RNA complementary to the genome appeared (Fig. 5) and no protein synthesis (Fig. 6). Furthermore, no defective interfering particles were produced by cells infected with subviral particles (not shown). This is quite consistent with the results observed by Moyer and Gatchell (1979), who showed that an important protein synthesis is present, even if RNA synthesis is very low, when cells are producing DI particles. Nevertheless, the rescue of VSV subviral particles by uv-irradiated virions was observed in CEC even in the presence of antiG antiserum (Table 3); this indicates again that subviral particles indeed penetrated into the cells (probably into the cytoplasm) and that some component(s) of the viral envelope of the parental virion is (are) necessary for developing an efficient viral cycle, probably at the beginning. Since efficiency or inefficiency of the rescue depended on the host cell used, host cell factor(s) could be involved in the onset of VSV transcription and/or replication. Our findings are in agreement with those published by Nowakowski et al. (1973), Szilagyi and Pringle (1975), Pringle (1978), Holland et al. (1976), Simpson et al. (1979), and Horikami and Moyer (1982). Furthermore, these results seem to indicate an interaction between the viral envelope components and the host cell factors involved in these processes. The cycle induced by subviral particles caused very little change in the host cells: (i) No inhibition of cell protein synthesis

ET AL.

was detected (Fig. 6), whereas a normal VSV infection rapidly produces such an inhibition, as was previously shown (Genty and Berreur, 1970). (ii) No morphological change was observed, whereas infection by normal virion quickly leads to a more or less pronounced alteration of infected cells, depending on the host cells considered (Baxt and Bablanian, 1976; Marcus and Sekellick, 1974; Genty and Bussereau, 1980). (iii) In CEC, uridine uptake was not inhibited following infection by subviral parI.icles, whereas after infection by virions uridine phosphorylation was inhibited even at low m.o.i., even in the presence of cycloheximide (Genty, 1975, Genty and Dragun, 1983). Therefore, it is likely that the G protein and perhaps some other VSV envelope component(s) is (are) involved in the modification of cell metabolism after infection by VSV virions. The finding of Genty (1975) that the M protein is involved in the uridine uptake could result from a direct effect as well as from an interaction of the two VSV envelope proteins. Their presence is also necessary to induce modifications of the host cell metabolism; nevertheless it cannot be completely ruled out that virions and subviral particles are located at different cytoplasmic sites and, therefore, behave differently. ACKNOWLEDGMENTS This work was partially supported by grants from the Centre National de la Recherche Scientifique (L.A. 136 and L.A. 040086). We thank Liliane Chamaillard, Yvonne Bouvet, Jacqueline Benejean, Chantal Thiers, and Josette Gagnat for their excellent technical assistance; and Gisele Bogdanoff for typing the manuscript. We are very grateful to R. Salesse for carrying out the lipid analysis.

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