Defective vaccinia virus particles in interferon-treated infected cells

VIROLOGY 133,220-227

(1984)

Defective Vaccinia Virus Particles in Interferon-Treated MARIANO

Infected Cells

ESTEBAN'

Departments of Biochemistry, Micrti~, and Immunology, SUNY, Downstate Medical Genter, Brooklyn, New York 1120$ Received June 22, 1989;accepted December 19, 1982 Vaccinia virus particles formed in interferon-treated, infected cells have been isolated. These particles have been characterized with regard to polypeptide composition, and ability to adsorb, penetrate, uncoat, and synthesize proteins in infected cells. As determined by one-dimensional SDS-PAGE analysis, with interferon concentrations of 100-500 u/ml, the pattern of [?S]methionine-labeled virion proteins was not altered; higher doses of interferon resulted in decreased labeling of some proteins. However, interferon doses of 100-500 u/ml decreased phosphorylation of vaccinia virus basic core polypeptide (P-11) by 30-70s; the same doses of interferon decreased the labeling of virus glycoproteins by 40-80%. Virus purified from interferon-treated cells adsorb, penetrate, and uncoat to a lesser extent than virus control. During infection to cells, these virus particles caused shutoff and synthesized the same spectrum of viral proteins as normal virus. These findings show that there are protein alterations in vaccinia virus particles isolated from interferon-treated, infected cells. These alterations may contribute to limit the spread of virus infection.

Interferons have many biological effects on animal cells (1). They are antiviral (Z4), inhibit cell growth (5), and regulate the immune system (6). The first two effects correlate with changes in the plasma membrane of treated cells (7, 8) and with the induction of specific enzymes (2, 3). These may lead to a block in the maturation and release of certain viruses or to a block at the level of gene expression of other viruses (l-8). Since the terminal step in enveloped virus maturation is budding from the cell surface, changes in the plasma membrane by interferon may account for inhibition of virus spreading between cells. A decreased amount of glycoprotein and membrane protein has been observed in VW released from treated cells. Here, inhibition of an early step in the formation of asparagine-linked oligosaccharide chains was found (9,10). In cells

1 Author addressed.

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infected with retroviruses, interferon treatment had no effect on viral RNA, reverse transcriptase, or protein synthesis. However, virus production was reduced, presumably as a result of changes in the plasma membrane of the cells (7, 8). Vaccinia virus is an enveloped virus that multiplies in the cytoplasm of infected cells where most of the virus remains cell associated (11,12). Interferon has been shown to inhibit vaccinia virus protein and DNA synthesis, block viral uncoating, and enhance significantly early viral RNA synthesis (B-17). This report addresses the question of whether interferon has any effect on the intracellular vaccinia virus that bypasses the early block. Mouse interferon, a mixture of a! (20%) and P (80%) species (sp act of 4 X lo7 units/ mg protein, kindly provided by the late K. Paucker), was titrated with VSV as a challenge virus against a mouse interferon reference standard (G002-904-511) from the Antiviral Substances Program, NIH. The titers are given as international reference 220

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units throughout. Mouse L cells (15) growing as monolayers in 150-mm dishes were treated with various concentrations of mouse interferon for 18 hr at 37” in MEM medium containing 10% heat-inactivated newborn calf serum (NCS). Thereafter, medium was removed and cells infected with purified vaccinia virus at a multiplicity of infection of 250 particles per cell (5 PFU/cell) in 5 ml of MEM. After 1 hr of virus adsorption at 37”, the inoculum was removed, and 20 ml of fresh medium with 1% dialyzed fetal calf serum added. Using [3H]thymidine-labeled vaccinia virus, and under conditions where overall protein synthesis was blocked with cycloheximide, no differences were seen in the number of virions that adsorb, penetrate, and form cores in untreated and IFNtreated, infected cells (not shown). This type of analysis was carried out by velocity sedimentation in sucrose gradients of cytoplasmic cell extracts (19). Interferon did not alter the rate of formation of cores, as it was observed previously (14). At 2 hr post infection (p.i.), cells were labeled independently with 10 pCi/ml [35S]methionine (1000 Ci/mmol), 10 &Vrnl [32P]orthophosphate, 10 pCi/ml PHfglucosamine (22,6 Ci/mmol), or 10 $Ji/m$H]thymidine (55 Ci/mmol). Depending on the experiment

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the medium contained 10% of the normal levels of methionine, phosphate, or glucose, respectively. At 20 hr p.i., cells were collected by scraping and all subsequent operations carried out at 4”. They were centrifugated, washed 3~ in PBS, resuspended in 1 ml of 1 mlM Na2HP04, and sonicated 3 x 5 seconds each in a sonifer (Biosonik) at 50 W. Cell extracts were centrifuged at 2K/lO min, the supernatants collected, and the pellet reextracted as previously. Pooled supernatants were spun at 15K/30 min in a Sorvall RCZ-B, the pellet resuspended by sonication (2 X 5 seconds) in 1 ml of 1 mM Na2HP04, applied over a cushion of 45% sucrose, and spun at 20K/30 min in a Beckman L2-50, SW-50 rotor. The pellet was resuspended by sonication (2 X 5 seconds) in 1 mM N%HPO*, applied over 20-45s (w/v) sucrose gradient in the same solution and centrifuged at 15K/20 min. The virus band was collected, diluted in 1 mM Na2HP04, sedimented at 15K/30 min in a Sorvall, and the virus pellet resuspended in a small volume of 1 mlM Na2HP04. The optical density at 260 mm was measured and the virus was aliquoted and stored at -20” for subsequent analysis. Protein concentrations were determined by the method of Bramhall et al (18). Table 1 shows the virus yields and specific activ-

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VIRUS YIELDS AND SPECIFIC ACXIVITIES OF VACCINIA VIRUS FROM UNTREATED AND IFN-TREATED, INFECTED CELLS Titration (cell extracts) (PFU X 109) Virus Untreated control IFN-treated (100 u/ml) IFN-treated (500 u/ml) IFN-treated (1000 u/ml)

$S]WR 4.2 2.6 2.6 2.5

[?lwR 7.0 5.6 4.2 4.0

Purified rH]WR 2.8 2.6 1.4 1.0

[%]WR 33,842 31,915 24,456 24,050

virus (cpm/pg) [=P]WR

[3H]WR

6687 5936 6095 5172

445 379 353 342

Note. Mouse L cells in X%-mm dishes were treated with various concentrations of mouse interferon, infected with vaccinia virus, labeled with radioactive precursors, and virus purified from cell extracts as indicated in the text. Virus titration in cell extracts was carried out with Vero cells. The specific activities (cpm/gg protein) of purified virus are given.

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FIG. 1. SDS-PAGE analysis of [%S]methionine, “P, and [sHlglucosamine-purifed labeled vaccinia virus particles obtained from untreated and interferon-treated, infected cells. Equal number of cpm was analyzed per sample in 10-X% gradient gel. Numbers on the top denote the origin of the labeled virus as well as the interferon concentrations. Molecular weight markers are included. The gel was impregnated with NEN enhancer, Molecular weights refer to the following markers: 14,300 lysozyme; 34,000 trypsinogen; 34,700 pepsin; 45,000 ovalbumin; 66,600 bovine plasma albumin. (A) Autoradiograms. (B) Densitometric analysis of the autoradiograms of virion proteins from untreated and interferon-treated (100 and 500 u/ml), infected cells.

ities of vaccinia virus preparations from untreated and IFN-treated, infected cells. The relative purities of the labeled virus preparations were similar as determined by sucrose gradient analysis and by electron microscopy (not shown).

In order to test if the polypeptide composition of vaccinia virus was changed by interferon, the labeled virion proteins were analyzed by SDS-PAGE followed by autoradiography. As presented in Fig. IA, the [35S]methionine-containing proteins

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FIG. l-Continued

were similar in virus particles purified from untreated or interferon-treated, infected cells. There was a reduction in some viral proteins (i.e., P24) with the highest interferon dose. When proteins from 32Plabeled virions were examined, it was found that in interferon-treated infected cells the major phosphoprotein component of a molecular weight of about 11,000 Da (11, 12) was by far the most inhibited of the several labeled bands. This inhibition was dose dependent. Similarly, after analysis of the virus glycoproteins, it was clear that glycosylation was greatly reduced in preparations from interferon-treated, infected cells. No differences were seen in the relative amount of proteins after staining

with Coomassie blue for any virus preparation. Figure 1B shows a densitometric analysis of the autoradiograms. Interferon doses of 100-500 u/ml decreased phosphorylation of vaccinia virus basic core polypeptide (P-11) by 30-7076; the same doses of interferon decreased the labeling of virus glycoproteins by 40-80%. Since vaccinia virus contains over 100 different polypeptides (1.2) the intensity of bands in the autoradiograms demonstrate only semiquantitative differences. Alterations in protein modification o:f vaccinia virus structural polypeptides may lead to particles with defects in replication. Therefore, we examined several stages of the vaccinia virus replication cycle: ad-

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EXTENT OF VACCINIA VIRUS ADSORPTION AND INFECTIVITY Adsorption Virus

[%S]WR

Untreated controls IFN-treated (100 u/ml) IFN-treated (500 u/ml) IFN-treated (1000 u/ml)

100 66 63 56

(% of control) [=P]WR 100 68 55 46

Infectivity

(% of control)

[3H]WR

[=S]WR

[=‘P]WR

[3H]WR

100 62 53 42

100 60 58 58

100 80 60 57

100 72 54 38

Note. Virus adsorption was measured in 24 Limbro wells of confluent mouse L cells infected with 1000 particles per cell of purified labeled virions obtained from untreated and interferon-treated, infected cells. After 1 hr of virus adsorption in 0.1 ml MEM, monolayers were washed 4X 1.0 ml PBS, then 5% TCA was added for 1 hr, washed in ethanol, dried, dissolved in 0.2 ml 0.3 NNaOH, 0.5% SDS, and radioactivity counted on Aquasol. Results are expressed as percentage of the radioactivity obtained from control-labeled virions and are the average of three independent determinations. Virus infectivity was determined by a virus plaque assay on BSC-40 monkey kidney cells plated in 60-mm dishes. Triplicates were infected with a series of dilutions of purified virus to give an equivalent of 100-309 PFU per dish. The same number of virus particles, as measured by optical density and protein content, was added to cells for each virus preparation in 0.5 ml MEM. After 1 hr of virus adsorption, inoculum was removed and cells overlayed with 5 ml of medium containing 0.9% noble agar and 2% NCS. After 3-5 days of incubation at 37” in a humidified atmosphere containing 5% COz, the agar overlay was removed and plaques counted after staining in 1% crystal violet with 2% ethanol. Results are expressed as percentage of the number of virus plaques obtained from controllabeled virions.

sorption, penetration, uncoating, and protein synthesis in infected cells. The degree of virus adsorption of 35S-, 32P- and 3H-labeled virus was monitored by determining the amount of the radioactive virus that remained cell associated during infection as well as by virus plaque assay. The results presented in Table 2, showed that the amount of labeled virus that remained cell associated was reduced by interferon. Moreover, infectivity was reduced. When equivalent numbers of virus particles were added to monolayer cultures of monkey cells (BSC-40) growing in 60mm dishes, the inoculum was removed after 1 hr of virus adsorption, cultures were overlayed with agar, and plaques were scored after 3-5 days of incubation at 3’7”, a reduction (50%) in the number of plaques was observed with purified virus from interferon-treated, infected cells. This reduction in both cell attachment and virus infectivity was not dependent on the cell

type but was species specific, since virus grown in Hela cells treated with mouse interferon, produced a normal virus (data not shown). To study virus penetration and uncoating, monolayer cultures of L cells in lOOmm dishes were infected for 60 min at 3’7” with 1 ml of MEM medium containing 32P or rH]thymidine-labeled purified virus which was added usually at 1000-2000 particles/cell. After incubation, the inoculum was removed and the cells were washed 3 X 10 ml with MEM. Then the monolayer was incubated with 10 ml of MEM + 2% NCS containing 100 pg/ml of cycloheximide. This drug inhibits protein synthesis, and can be conveniently used to measure the extent of conversion of vaccinia virus particles to subviral cores (19). At 4 hr p.i., the cell monolayers were washed 3~ in PBS, scraped, collected by centrifugation, resuspended in 1.0 ml PBS containing 1% Nonidet-P40, and after 10 min on ice

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FIG. 2. Sucrose sedimentation analysis of the extent of vaccinia virus penetration and uncoating in mouse L cells. “P-labeled purified virus were obtained from untreated (A), and interferon-treated at 500 units/ ml (B) or 1000 units/ml (C), infected cells. The multiplicity of virus infection used was 2000 particles per cell. Sedimentation is from right to left. The fast sedimenting peak of radioactivity corresponds to a virus particle followed by a subviral core (19). The percentage of total radioactivity in cpm recovered in

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dounced 10X. The nuclei were removed by centrifugation (2K/lO min), supernatants collected, sonicated 3 X 5 set each, applied over a 20-45s w/v sucrose gradient in 1 mM NazHP04, and centrifuged at 15K/20 min in, SW-41 rotor (Beckman), 4”. The fractions were collected from the bottom of the tube with a pump, TCA precipitated, and the radioactivity was determined by liquid scintillation counting. As indicated in Fig. 2 for 32P-labeled virus, in cells infected with normal virus, two peaks of radioactivity were found corresponding to a virus particle and a subviral core (19). Protein remained on the top of the gradient. In cells infected with purified virus obtained from interferon-treated cells there was a marked decrease in the amount of both virus particles. Similar results were obtained with r3H]thymidine virus (not shown). The degree of viral protein synthesis in infected cells was determined after pulse labeling with r5S]methionine followed by SDS-PAGE and autoradiography. As indicated in Fig. 3, virus isolated from interferon-treated cells inhibited host protein synthesis and synthesized the same spectrum of viral proteins in infected cells as did virions from control cells. Densitometric analysis of the autoradiograms revealed a reduction in the relative amounts of some viral proteins in cells infected with virions from interferon-treated cells. In conclusion, the results presented in this report show that (a) matured vaccinia virus particles are produced in interferontreated, infected cells, (b) these particles have defects in protein modification, (c) when in contact with cells, these particles adsorb, penetrate, and uncoat to lesser extent than virus control, (d) in the course of virus infection, these particles caused shutoff and synthesized similar viral proteins as normal virus. virus particle (fractions 8-12) and subviral core (fractions 13-17) were, respectively, 21 and 13% (virus control); 11 and 7% (virus from interferon-treated (1000 u/ml) cells).

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FIG. 3. Protein synthesis in cells infected with vaccinia virions from interferon-treated cells. Cultures of mouse L cells grown in tissue culture plates (24 wells) were infected with 1000 particles/ cell of purified vaccinia virus. At 3 hr pi cells were pulse labeled with 10 pCi/ml of [%]methionine for 2 hr. Thereafter, cell extracts were prepared and the same amount of protein from each culture was analyzed by SDS-PAGE. (A) Autoradiogram of proteins synthesized in uninfected cells (U), cells infected with normal virus (0) and cells infected with virus obtained from interferon-treated (1000 u/ml) cells. (B) Densitometric analysis of the autoradiogram for infected cells. Similar results as (A) and (B) were obtained using pS]WR and PHjWR virions. (C) Extent of protein synthesis in interferon-treated (10 and 100 u/ml) cells, infected with 20 PFU/cell of VSV for 5 hr. Molecular weights of some of the vaccinia polypeptides are indicated.

ACKNOWLEDGMENTS This investigation was supported by Public Health Service Grant ROI AI 16780 from the National Institute of Allergy and Infectious Diseases and by a New York State Health Research Council Award. I thank J. A. Lewis and R. Bablanian for critically reading the manuscript and Victoria Jimenez for her skilled technical assistance and graphic art. REFERENCES 1. STEWART, W. E., II, “The Interferon Springer-Verlag, New York/Berlin,

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