Interferon-treated cells release vesicular stomatitis virus particles ...

Proc. Nati. Acad. Sci. USA Vol. 77, No. 4, pp. 2284-2287, April 1980 Microbiology

Interferon-treated cells release vesicular stomatitis virus particles lacking glycoprotein spikes: Correlation with biochemical data (infectivity/interferon action/noninfectious virus particles)

RADHA K. MAHESHWARI, ANTHONY E. DEMSEY, SASHI B. MOHANTY*, AND ROBERT M. FRIEDMAN Laboratory of Experimental Pathology, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20205; and *Department of Veterinary Science, University of Maryland, College Park, Maryland 20742

Communicated by Bernhard Witkop, January 10, 1980

ABSTRACT Earlier we reported a reduction to 1/30th1/100th of the original number of infectious particles in the infectious vesicular stomatitis virus (VSV) released from L cells treated with 10 or 30 reference units of interferon per ml. However, in these cultures virus particle production, as measured by VSV particle-associated viral RNA, virus nucleocapsid protein, and viral transcriptase, was inhibited by less than 10%. Data reported in this paper show that there was a significant reduction in glycoprotein and membrane protein of VSV particles released from interferon-treated cells. Evidence supporting the deficiency of glycoprotein in VSV released from interferon-treated cells was derived from electron microscopic studies. Under conditions where glycoprotein spikes or projections were clearly detectable on the surface of VSV released from cells not treated with interferon, very few spikes were observed on VSV released from interferon-treated cells. These results suggested that interferon-treated cells produced VSV particles with low infectivity and that this low infectivity may be related to the reduced amount of glycoprotein and membrane protein incorporated into such particles. Interferon inhibits the replication of a wide variety of viruses. In most systems studied, virus-directed translation or transcription was inhibited (1); however, an unusual inhibitory effect of interferon treatment on the replication of RNA tumor viruses has been reported. Inhibition of RNA tumor virus production was not correlated with inhibition of any of the intracellular steps in virus replication that were tested (2-6). Inhibition of virus production was not correlated with inhibition of the accumulation of viral RNA (7) or of viral proteins (8). In interferon-treated AKR cells there was decreased production of both endogenous murine leukemia virus (MLV) particles and infectious MLV; however, the intracellular concentration of viral p30 antigen was increased (8). Studies on the action of interferon in Moloney MLV infection of mouse bone marrow/thymus (TB) cells showed that in interferon-treated cells there was a decrease in the production of infectious MLV to 1/2000th the original number, a decrease in the level of virus-specific reverse transcriptase activity to 1/lOth-1/20th the original activity, and only a 2-fold difference in the number of budding viral particles observed on the plasma membrane as determined by scanning electron microscopy studies (9). These results suggested that in these systems, either the release of virus from the plasma membrane or the production of infectious virus particles was inhibited by treatment with interferon. One important question about this work is whether such findings are restricted to RNA tumor virus systems. We have studied the effect of low concentrations of interferon (10 or 30 reference units/ml) on vesicular stomatitis virus (VSV) infection of L cells in contrast to previous studies, which used much The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate

higher interferon concentrations and reported inhibition of VSV-directed translation or transcription (10-11). We observed (12) that in L cells treated with 10 or 30 reference units of interferon per ml, there was a decrease in the prqduction of infectious VSV to 1/3Oth-1/200th the original number; virus particle production, as measured by VSV particle-associated viral RNA, virus nucleocapsid protein, and virus transcriptase, was inhibited by a maximum of 10%. We now report a significantly greater reduction in glycoprotein (G) and membrane (M) protein of VSV released from interferon-treated cells. These results suggested that interferon-treated cells produce VSV particles with low infectivity because of the reduced amount of G and M proteins incorporated into such particles. Evidence in support of the reduction in viral glycoprotein in the envelope of virus from interferon-treated cells was derived from electron microscopic studies. Under conditions such that the spikes or projections were clearly detectable on the surface of wild-type VSV, very few spikes were observed on VSV released from interferon-treated cells. MATERIALS AND METHODS Virus Stock and Virus Assays. VSV was the Indiana strain originally obtained from C. Buckler (National Institute of Allergy and Infectious Diseases, Bethesda, MD). It had been plaque-purified and passaged by us at a multiplicity of 0.01 in Vero cells. No defective interfering particles could be detected in our stock of virus. The pool of virus used was assayed by plaque titration in Vero cells, where it titered at 2 X 109 plaque-forming units (PFU)/ml. Cells. L cells were originally obtained from D. Burke (Department of Biological Sciences, University of Warwick, Coventry, England). They were grown in monolayers in Eagle's minimal essential medium supplemented with glutamine (0.03 g/500 ml), 10% (vol/vol) heat-inactivated fetal calf serum (GIBCO), penicillin (100 units/ml), streptomycin (100 ,gg/ml), Fungizone (0.25 tg/ml), and gentamycin (5 ,tg/ml). These cells are very sensitive to the antiviral activity of mouse interferon. Vero cells were obtained from the National Institutes of Health media unit and were also grown in Eagle's medium supplemented with 10% fetal calf serum. Interferon and Interferon Assays. Mouse interferon was prepared and partially purified on an antibody affinity column (13) in the laboratory of K. Paucker (Pennsylvania College of Medicine, Philadelphia, PA). The specific activity of the preparations used was at least 2 X 107 mouse reference units per mg of protein. The antiviral activity of this preparation had the physical and chemical properties usually ascribed to interferon. The interferon was assayed by plaque reduction assay Abbreviations: VSV, vesicular stomatitis virus; MLV, murine leukemia virus; G protein, glycoprotein; M protein, membrane protein; PFU, plaque-forming units.

this fact.

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in L cells with VSV or by cytopathic effect reduction with encephalomyocarditis virus. Preparation and Purification of Virus Samples. L cells were grown in 150-cm2 flasks to confluence (2 X 107 cells) and treated with 0-30 units of interferon per ml for 14 hr; the cell monolayers were washed three or four times to remove residual interferon. All the monolayers were then infected with VSV at a multiplicity of 5 PFU/cell; virus was adsorbed for 45 min. The unadsorbed virus was removed and the cells were refed with 15 ml of Eagle's medium and incubated for 16 hr. The supernatant fluids were then collected and centrifuged at 2000 X g to remove cell debris. Aliquots of each sample were saved for assay of virus infectivity and electron microscope studies; the remaining supernatant fluids were centrifuged at 48,000 X g for 2 hr. The pellet was resuspended in a small volume of TES buffer (50 mM Tris-HCI, pH 7.2/0.1 M NaCl/1 mM EDTA), and an aliquot of each sample was saved for electron microscope studies. The pelleted virus was further purified in a 20-50% (wt/wt) equilibrium sucrose gradient in TES buffer by centrifuging at 72,000 X g for 16 hr in an SW27 rotor. The virus that banded at a density of 1.16-1.18 g/ml was sedimented for 3 hr at 110,000 X g. The pellet containing the purified virus was resuspended in 0.1 M NaCl/10 mM sodium acetate buffer (pH 7.2). Analysis of Virion Proteins. Radiolabeled VSV (14) was prepared as follows: L cells were treated with interferon (30 units/ml) as above. After virus adsorption, the monolayers were washed with leucine-free medium and then [3H]leucine (20 11Ci/ml, specific activity 121.1 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels; New England Nuclear) was added in special Eagle's medium devoid of unlabeled leucine and serum. After incubation for 8 hr at 37°C, 5 ml of Eagle's medium with 2% dialyzed fetal calf serum was added to each culture. The supernatant fluids were collected at 16 hr and virus was purified by banding on a sucrose gradient as described above. Virion proteins were analyzed by NaDodSO4/polyacrylamide gel electrophoresis (14). After electrophoresis, the gels were stained and dried, and the incorporation of radioactive precursor was quantitated by fluorography (15). Negative Staining of Virus for Electron Microscope Studies. Droplets of viral suspensions on Formvar carboncoated grids were negatively stained with 4% sodium phosphotungstate at pH 6.5. Similar results were obtained with lower concentrations of phosphotungstate. Unfixed suspensions were stained directly, whereas preparations fixed with 5% glutaraldehyde were washed once with distilled water before staining. All preparations were air-dried and examined with a Hitachi HU-11 electron microscope operating at 75 kV.

Proc. Natl. Acad. Sci. USA 77 (1980)

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Table 1. Effect of interferon treatment of L cells on production of VSV Interferon, units/ml

Virus titer, PFU/ml

Inhibition, loglo

0 6X107 3 X 106 1.2 10 2.0 30 5x 105 L cells were treated with 0-30 units of interferon per ml for 14 hr, followed by washing of the monolayers three or four times to remove residual interferon. Monolayers were challenged with VSV at a multiplicity of 5 PFU/cell and incubated for 16 hr. The virus assays in these fluids were carried out by using the plaque titration method in Vero cells.

nally, their precipitation in similarly conducted and concurrent studies with antiserum to VSV. Four proteins were clearly detected in samples of control virus; however, in virus from interferon-treated cells, there was a marked decrease in the concentration of G and M proteins. The migration of proteins was similar in both the samples under the present experimental conditions. The gels were scanned in a densitometer (Helena Laboratories, Beaumont, TX). These scans demonstrated the selective inhibition in G and M protein in the VSV released from cells treated with 30 units of interferon per ml (Fig. 1). Electron Microscopy. After the observations that VSV particles released from interferon-treated cells were deficient in G and M proteins, we examined the morphology of these particles. As suggested by the results of analysis of proteins on

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RESULTS Assay of Virus Infectivity in Samples from Cells Treated

with Interferon. Supernatant fluids were assayed for virus by a plaque titration method with Vero cells. Data (Table 1) indicated that the inhibition in virus yield was directly related to the concentration of interferon used. With 30 units of interferon per ml, a decrease in titer of infectious virus to 1/100th the original number was observed. However, as previously reported (12), virion-associated RNA, transcriptase activity, and nucleocapsid (N) protein were decreased by less than 10% in virus produced by interferon-treated cells. Effect of Interferon on VSV Proteins. The same amount of radioactivity (10,000 cpm) in virus samples from control and interferon-treated cells was applied to slab gels and the radioactivity was quantitated by fluorography. Proteins were identified as being viral on the basis of their molecular weight, their comigration with the proteins of purified VSV, and, fi-

DISTANCE MIGRATED

P

FIG. 1. Fluorograms and densitometer scans of VSV proteins from purified virus. Equal amounts of radioactivity (10,000 cpm) were applied to each gel. The gels were electrophoresed and then stained with Coomassie brilliant blue R. After the gels were destained they were impregnated with 2,5-diphenyloxazole (New England Nuclear), dried, and exposed to Kodak X-OMAT x-ray film. Phosphorylase B (Mr 92,500), bovine serum albumin (Mr 69,000), ovalbumin (Mr 46,000), carbonic anhydrase (Mr 30,000), and cytochrome c (Mr 12,300) were used as reference protein molecular weight markers. (a) Proteins of VSV released from cells treated with interferon (30 reference units/ml); (b) proteins of VSV released from cells not treated with interferon. N, nucleocapsid protein.

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Proc. Natl. Acad. Sci. USA 77 (1980) Microbiology: Maheshwari et al.PrcNaiAadSc.UA7(90 J..

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a

b

d

h

FIG. 2. Electron micrographs of negatively stained (4% phosphotungstic acid) VSV. Confluent L-cell monolayers were treated with interferon (0-30 units/ml) for 14 hr and cultures were washed. The cells were then infected with VSV at 5 PFU/cell and incubated for 22 hr. The supernatant fluids containing released VSV particles were processed as follows: 20 ml of supernatant was layered on a 10-ml equilibrium sucrose gradient (15-50%, wt/wt) and centrifuged in an SW27 rotor at 82,000 X g for 16 hr. The fractions containing virus were dialyzed against Dulbecco's phosphate-buffered saline to remove sucrose and samples were examined. (X195,000.) (a) VSV released from cells not treated with interferon; (b VSV released from cells treated with interferon (10 units/ml); (c and d) VSV released from cells treated with interferon (30 units/ml). (e-g) Other supernatant fluids were concentrated 100-fold by pelleting at 72,000 X g for 90 min and observed. (X170,000.) (e) VSV released from cells not treated with interferon; (f and g) VSV released from cells treated with interferon (30 units/ml). (h-k) Additional supernatants were fixed with 1/3 vol of glutaraldehyde (5%) in Dulbecco's phosphate-buffered saline at 4 C and examined. (X170,000.) (h and i) VSV released from cells not treated with interferon; (j and h) VSV released from cells treated with interferon (30 units/ml).

gels, electron microscopic observations clearly revealed very few glycoprotein spikes present on the surface of VSV released from interferon-treated cells. (Fig. 2 b-d, f, gj and k); under

the same conditions the spikes or surf-ace projections were clearly detectable on VSV released from cells not treated with interferon (Fig. 2 a, e, h, and i). The reduction of spikes in a

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Maheshwari et al.

virus preparation was dependent on the concentration of interferon used to treat the cells. With 10 units of interferon per ml, we could see a few spikes (Fig. 2 b); however, with 30 units of interferon, spikes were quite scarce (Fig. 2 c, d, f, g, j, and k).

DISCUSSION Our previously reported results indicated that in VSV released from interferon-treated cells, there was a marked reduction in VSV infectivit.y compared to virion transcriptase, virion nucleoprotein, or virion RNA content (12). Furthermore, VSV particles produced in interferon-treated cells appeared to contain only 42S viral RNA. They did not interfere with the growth of wild-type VSV nor did they produce interferon in culture. Such particles are, therefore, unlikely to be defective interfering forms of VSV. We have observed that VSV particles released from interferon-treated cells were very deficient in G and M protein. Evidence supporting the reduction of viral glycoprotein from the viral envelope is further strengthened by electron microscope studies (Fig. 2). These data together suggested that in the presence of interferon not only was the production of infectious virus particles inhibited, but also the production of noninfectious virus particles was disproportionately increased. Previous investigations on the ultrastructure of VSV had demonstrated the presence of spikes on the surface of wild-type virus particles when examined by negative staining techniques (16). The identity of the glycoprotein of VSV as the spike protein has been confirmed by treatment of these particles with proteases such as Pronase, which digested the surface proteins without rupturing the lipid bilayer; this resulted in the removal of the spikes with the concomitant production of noninfectious viral particles (16). The production of noninfectious virus particles morphologically and biochemically differing from wild-type particles has been reported in group V temperature-sensitive (ts) mutants of VSV grown at nonpermissive temperatures and also with some mutants of Rous sarcoma virus (17-21). In several instances the production of noninfectious particles was associated with absence of major glycoproteins. These data suggest that at least some of the reduced infectivity of the VSV particles released from interferon-treated L cells may well be related to the reduced amount of glycoprotein incorporated into such particles. It is possible that interferon-induced changes in the cell plasma membrane (22-24) may be the basis for the alteration in infectivity of both VSV and murine leukemia virus produced

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after treatment with interferon because the morphogenesis of these viruses involves budding from the cell surface. We thank F. Mottram for able technical support and Dr. D. Epstein for many helpful discussions and review of the manuscript.

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