Delayed appearance of pseudotypes between vesicular stomatitis virus influenza virus during mixed infection of MDCK cells.

In intact Madin-Darby canine kidney (MDCK) cell monolayers, vesicular stomatitis virus (VSV) matures only at basolateral membranes beneath tight junctions, whereas influenza virus buds from apical cell surfaces. Early in the growth cycle, the viral glycoproteins are restricted to the membrane domain from which each virus buds. We report here that phenotypic mixing and formation of VSV pseudotypes occurred when influenza virus-infected MDCK cells were superinfected with VSV. Up to 75% of the infectious VSV particles from such experiments were neutralized by antiserum specific for influenza virus, and a smaller proportion (up to 3%) were resistant to neutralization with antiserum specific for VSV. The latter particles, which were neutralized by antiserum to influenza A/WSN virus, are designated as VSV(WSN) pseudotypes. During mixed infections, both wild-type viruses were detected 1 to 2 h before either phenotypically mixed VSV or VSV(WSN) pseudotypes. Coincident with the appearance of cytopathic effects in the monolayer, the yield of pseudotypes rose dramatically. In contrast, in doubly infected BHK-21 cells, which do not show polarity in virus maturation sites and are not connected by tight junctions, VSV(WSN) pseudotypes were detected as soon as VSV titers rose to the minimum levels which allowed detection of pseudotypes, and the proportion observed remained relatively constant at later times. Examination of thin sections of doubly infected MDCK monolayers revealed that polarity in maturation sites was preserved for both viruses until approximately 12 h after inoculation with influenza virus, when disruption of junctional complexes was evident. Even at later periods, the majority of each virus type was associated with its normal membrane domain, suggesting that the sorting mechanisms responsible for directing the glycoproteins of VSV and influenza virus to separate surface domains continue to operate in doubly infected MDCK cells. The time course of VSV(WSN) pseudotype formation and changes in virus maturation sites are compatible with progressive mixing of viral glycoproteins at either intracellular or plasma membranes of doubly infected cells.     

The M protein of vesicular stomatitis virus associates specifically with the basolateral membranes of polarized epithelial cells independently of the G protein

Using monoclonal antibodies and indirect immunofluorescence microscopy, we investigated the distribution of the M protein in situ in vesicular stomatitis virus-(VSV) infected MDCK cells. M protein was observed free in the cytoplasm and associated with the plasma membrane. Using the ts045 mutant of VSV to uncouple the synthesis and transport of the VSV G protein we demonstrated that this distribution was not related to the presence of G protein on the cell surface. Sections of epon-embedded infected cells labeled with antibody to the M protein and processed for indirect horseradish peroxidase immunocytochemistry revealed that the M protein was associated specifically with the basolateral plasma membrane. The G and M proteins of VSV have therefore evolved features which bring them independently to the basolateral membrane of polarized epithelial cells and allow virus to bud specifically from that membrane.     

Inhibition of pinocytosis in rat embryo fibroblasts treated with monensin

Rat embryo fibroblasts cultured in the presence of monensin exhibited an inhibited uptake of horseradish peroxidase. The inhibition was detected after 3 h, after which time the cells became increasingly vacuolated; the concentration of monensin required to inhibit pinocytosis (0.4 microM for half-maximum inhibition at 18 h) was similar to that found by others to inhibit secretion. Both the exchange of 5'-nucleotidase between the membranes of cytoplasmic organelles and the cell surface and the internalization of anti-5'-nucleotidase bound to the cell surface were inhibited by approximately 90% in monensin- treated cells. The effects of monensin were reversible: cells cultured first with monensin, and then in fresh medium, exhibited control levels of horseradish peroxidase uptake, exchange of 5'-nucleotidase, and internalization of anti-5'-nucleotidase bound to the cell surface. After monensin treatment, the median density of both galactosyl transferase and 5'-nucleotidase increased from 1.128 to 1.148, and the median density of both N-acetyl-beta-glucosaminidase and horseradish peroxidase taken up by endocytosis decreased from 1.194 to 1.160. The results indicate that monensin is a reversible inhibitor of pinocytosis and, presumably, therefore, of membrane recycling. They suggest that the inhibition of membrane recycling occurs at a step other than the fusion of pinocytic vesicles with lysosomes and is perhaps a consequence of an effect of the ionophore on the Golgi complex.     

Vesicular stomatitis virus mRNA and inhibition of translation of cellular mRNA--is there a P function in vesicular stomatitis virus?

Infection of animal cells by vesicular stomatitis virus (VSV) results in inhibition of translation of cellular mRNA. We showed previously that, in BHK cells infected by the Glasgow isolate of VSV Indiana, this is due to competition during the initiation step of protein synthesis of viral and cellular mRNA for a constant, limiting number of ribosomes. We show here that infection of the same cells with the San Juan isolate of VSV resulted in a more rapid shutoff of host protein synthesis and that this was paralleled by a more rapid accumulation of viral mRNA. Extending our conclusion that shutoff is due to mRNA competition, we show further that the average size of polysomes translating viral and cellular mRNA was threefold smaller in cells infected by VSV San Juan than by VSV Glasgow, which, in turn, was about one-half that of uninfected cells. In all cases, cellular and viral mRNA's which encoded the same-sized polypeptides were found on the same-sized polysomes, a result indicating that the efficiency of translation of both types of mRNA's is about the same in the infected cell. Also, there was no preferential sequestration of viral or cellular mRNA's in ribonucleoprotein particles. Additional correlations between the levels of viral mRNA's and the inhibition of protein synthesis came from studies of three other wild-type VSV strains and also from studies with Vero and L cells. In particular, the rate of shutoff of L-cell protein synthesis after infection by any VSV isolate was slower than that in BHK cells, and this was correlated with a slower rate of accumulation of viral mRNA. VSV temperature-sensitive mutants which synthesized, at the nonper-missive temperature, no VSV mRNA failed to inhibit synthesis of cellular proteins. Stanners and co-workers (C. P. Stanners, A. M. Francoeur, and T. Lam, Cell 11:273-281, 1977) claimed that VSV mutant R1 inhibited synthesis of L cell protein synthesis less rapidly than did its parent wild-type strain HR. They concluded that this effect was due to a mutation in an unspecified VSV protein, “P.” We found, in both L and BHK cells, that R1 infection resulted in a slightly slower inhibition of cellular mRNA translation than did HR infection and that this was correlated with a slightly reduced accumulation of VSV mRNA. The level of VSV mRNA, rather than any specific VSV protein, appeared to be the key factor in determining the rate of shutoff of host protein synthesis.     

Inhibition of vesicular stomatitis virus infection by nitric oxide.

Inhibitory effects of nitric oxide (NO) on vesicular stomatitis virus (VSV) infection were investigated by using a VSV-susceptible mouse neuroblastoma cell line, NB41A3. Productive VSV infection of NB41A3 cells was significantly inhibited by an organic NO donor, S-nitro-N-acetylpenicillamine (SNAP), while the control compound N-acetylpenicillamine (NAP) had no effect. Survival rate of VSV-infected cells was greatly increased by the treatment with SNAP, while the NAP treatment did not have any effect. Adding SNAP 30 min prior to infection resulted in complete inhibition of viral production when a low multiplicity of infection (MOI) was used. Substantial inhibition of viral production was also obtained with treating cells 6 h earlier before infection with a higher MOI. Activating the neuronal NO synthase by treating cells with N-methyl-D-aspartate (NMDA) led to significant inhibition of viral production by cells infected at the three doses of virus tested (MOIs of 0.1, 1, and 5). The inhibitory effect of NMDA on viral infection was totally blocked by the NO synthase inhibitor N-methyl-L-arginine. However, adding hemoglobin, a strong NO-binding protein and thus an inactivator of NO activity, did not reverse the NMDA-induced inhibition of viral production, suggesting that NO might exert its antiviral effects inside the NO-producing cells. Collectively, these data support the anti-VSV effects of NO, which might be one of the important factors of natural immunity in controlling the initial stages of VSV infection in the central nervous system.     

Vesicular stomatitis virus growth in Drosophila melanogaster cells: G protein deficiency.

In cultured Drosophila melanogaster cells, vesicular stomatitis virus (VSV) established a persistent, noncytopathic infection. No inhibition of host protein synthesis occurred even though all cells were initially infected. No defective interfering particles were detected, which would explain the establishment of the carrier state. In studies of the time course of viral protein synthesis in Drosophila cells, N, NS, and M viral polypeptides were readily detected within 1 h of infection. The yield of G protein and one of its precursors; G1, was very low at any time of the virus cycle; the released viruses always contained four to five times less G than those produced by chicken embryo cells, whatever the VSV strain or serotype used for infection and whatever the Drosophila cell line used as host. Actinomycin D added to the cells before infection enhanced VSV growth up to eight times. G and G1 synthesis increased much more than that of the other viral proteins when the cells were pretreated with the drug; nevertheless, the released viruses exhibited the same deficiency in G protein as the VSV released from untreated cells. Host cell control on both G-protein maturation process and synthesis at traduction level is discussed in relation to G biological properties.     

Persistence of vesicular stomatitis virus in cloned interleukin-2-dependent natural killer cell lines.

We have investigated virus-lymphocyte interactions by using cloned subpopulations of interleukin-2-dependent effector lymphocytes maintained in vitro. Cloned lines of H-2-restricted hapten- or virus-specific cytotoxic T lymphocytes (CTL) and alloantigen-specific CTL were resistant to productive infection by vesicular stomatitis virus (VSV). In contrast, cloned lines of natural killer (NK) cells were readily and persistently infected by VSV, a virus which is normally highly cytolytic. VSV-infected NK cells continued to proliferate, express viral surface antigen, and produce infectious virus. Furthermore, persistently infected NK cells showed no marked alteration of normal cellular morphology and continued to lyse NK-sensitive target cells albeit at a slightly but significantly reduced level. The persistence of VSV in NK cells did not appear to be caused by the generation of temperature-sensitive viral mutants, defective interfering particles, or interferon. Consequently, studies comparing the intracellular synthesis and maturation of VSV proteins in infected NK and mouse L cells were conducted. In contrast to L cells, in which host cell protein synthesis was essentially totally inhibited by infection, the infection of NK cells caused no marked diminution in the synthesis of host cell proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of viral proteins from infected cells showed that the maturation rate and size of VSV surface G glycoprotein were comparable in L cells and NK cells. Nucleocapsid (N) protein synthesis also appeared to be unaffected in NK cells. In contrast, the viral proteins NS and M appeared to be selectively degraded in NK cell extracts. Mixing experiments suggested that a protease in NK cells was responsible for the selective breakdown of VSV NS protein. Finally, VSV-infected NK cells were resistant to lysis by virus-specific CTL, suggesting that persistently infected NK cells may harbor virus and avoid cell-mediated immune destruction in an immunocompetent host.     

Vesicular stomatitis virus G protein acquires pH-independent fusion activity during transport in a polarized endometrial cell line

Entry of vesicular stomatitis virus (VSV), the prototype member of the rhabdovirus family, occurs by receptor-mediated endocytosis. Subsequently, during traversal through the endosomal compartments, the VSV G protein acquires a low-pH-induced fusion-competent form, allowing for fusion of the viral membrane with endosomal and lysosomal membranes. This fusion event releases genomic RNA into the cytoplasm of the cell. Here we provide evidence that the VSV G protein acquires a fusion-competent form during exocytosis in a polarized endometrial cell line, HEC-1A. VSV infection of HEC-1A cells results in high viral yields and giant cell formation. Syncytium formation is blocked in a concentration-dependent manner by treatment with the lysosomotropic weak base ammonium chloride, which raises intravesicular pH. Virus release is somewhat delayed by treatment with ammonium chloride, but virus yields gradually reach those of control cells. In addition, inhibition of vacuolar H(+)-ATPases by treatment with bafilomycin A1 also inhibited cell to cell fusion without altering virus yields. Virions released from infected HEC cells were themselves not fusion competent, since viral entry required an active H(+)-ATPase and a low-pH-induced conformational change in the viral G protein. Thus, the conformation change leading to fusion competence during exocytotic transport is reversible and reverts during or after release of the virion from the infected cell.     

Comparative studies on cytopathic effects induced by vesicular stomatitis virus in two cell types.

Infection of mouse L cells with vesicular stomatitis virus (VSV) leads to an extensive cell fusion, while porcine kidney stable (PS) cells infected with VSV show only cell rounding. Therefore, comparative morphological studies on the infection of the two cell lines were carried out using a transmission or scanning electron microscope and an immunofluorescence microscope. PS cells infected with VSV contrasted to L cells infected with the same virus in the following two points; (1) the principal site of VSV maturation was the intracytoplasmic vacuolar membrane in PS cells and the plasma membrane in L cells. However, it was found that viral glycoprotein was present on the cell surface of infected PS cells; (2) the morphological changes at the cell surface of infected PS cells occurred much earlier and were severer than those at the cell surface of infected L cells. From these observations, we discuss the possibility that the surfaceembrane of PS cells is too sensitive to the VSV-induced cell damage to cause cell fusion.     

Mechanism of interferon action: inhibition of vesicular stomatitis virus replication in human amnion U cells by cloned human gamma-interferon. I. Effect on early and late stages of the viral multiplication cycle

The molecular basis of the inhibition of vesicular stomatitis virus (VSV) replication by pure recombinant gamma-interferon (IFN-gamma) in human amnion U cells was examined. A saturating concentration of IFN-gamma induced, at maximum, about a two log10 reduction in infectious VSV yield. The kinetics of induction of the antiviral activity by IFN-gamma were first order over the period of about 6-18 h, following a lag of about 3 h, after treatment with a saturating concentration of IFN-gamma. The relationship of the inhibition in VSV infectivity to the early and late events of the VSV multiplication cycle was investigated. IFN-gamma treatment had no detectable effect on the adsorption and penetration of VSV virions or on their uncoating to yield viral nucleocapsids. The polypeptides of adsorbed or uncoated VSV particles were neither preferentially degraded nor detectably altered in IFN-gamma-treated U cells, as compared to untreated U cells. Progeny virions isolated from IFN-gamma-treated U cells, although greatly reduced in number, were found to be equally as infectious as those isolated from untreated U cells. Progeny virions from IFN-gamma-treated cells also possessed the same composition of viral proteins as was observed for virions from untreated cells. These results suggest that conditions of IFN-gamma treatment sufficient to reduce the yield of infectious VSV progeny 100-fold do not detectably affect either the early or the late stages of the VSV multiplication cycle.     

Effect of Vesicular Stomatitis Virus Infection on the Histocompatibility Antigen of L Cells

When mouse L cells are infected for 22 hr with vesicular stomatitis virus (VSV), a ribonucleic acid-containing enveloped virus, greater than 70% of the major histocompatibility antigen (H-2), is no longer detectable by the method of inhibition of immune cytolysis. Infected cells prelabeled with (14)C-glucosamine also show a correspondingly greater loss of trichloroacetic acid-insoluble radioactivity than uninfected cells. The loss of H-2 antigenic activity is not due to the viral inhibition of host cell protein synthesis since cells cultured for 18 hr in the presence of cycloheximide have the same amount of H-2 activity as untreated controls. Also, cells infected with encephalomyocarditis virus, a picornavirus, show no loss of H-2 activity at a time when host cell protein synthesis is completely inhibited. VSV structural proteins associated in vitro with uninfected L-cell plasma membranes do not render H-2 sites inaccessible to the assay. Although antibodies may not combine with all the H-2 antigenic sites on the plasma membrane, anti-H-2 serum reacted with L cells before infection does not prevent a normal infection with VSV. H-2 activity can be detected in virus samples purified from the medium of infected L cells; this virus purified after being mixed with L-cell homogenates shows greater H-2 activity than virus purified after being mixed with HeLa cell homogenates. However, VSV made in HeLa cells shows no H-2 activity when mixed with L-cell homogenates.     

Assembly of enveloped viruses in Madin-Darby canine kidney cells: polarized budding from single attached cells and from clusters of cells in suspension

In confluent monolayers of the dog kidney epithelial cell line Madin-Darby canine kidney (MDCK) assembly of RNA enveloped viruses reflects the functional polarization of the cells. Thus, influenza, Sendai, and Simian virus 5 bud from the apical (free) surface, while vesicular stomatitis virions (VSV) are assembled at basolateral plasma membrane domains (Rodriguez-Boulan, E., and D.D. Sabatini, 1978, Proc. Natl. Acad. Sci. U.S.A., 75:5071-5075). MDCK cells derived from confluent monolayers by dissociation with trypsin-EDTA and maintained as single cells in spinner medium for 12-20 h before infection, lose their characteristic structural polarity. Furthermore, when these cells were infected with influenza or VSV, virions assembled in a nonpolarized fashion over most of the cell surface. However, when dissociated MDCK cells infected in suspension were sparsely plated on collagen gels to prevent intercellular contact and the formation of junctions, the characteristic polarity of viral budding observed in confluent monolayers was again manifested; i.e., VSV budded preferentially from adherent surfaces and influenza almost exclusively from free surface regions. Similar polarization was observed in cells which became aggregated during incubation in spinner medium: influenza budded from the free surface, while VSV was produced at regions of cell-cell contact. It therefore appears that in isolated epithelial cells attachment to a substrate or to another cell is sufficient to trigger the expression of plasma membrane polarity which is manifested in the asymmetric budding of viruses.     

Effect of monensin on the synthesis, maturation and secretion of vesicular stomatitis virus proteins in a monensin-resistant Chinese hamster ovary cell line

We compared the effects of the cationic ionophore, monensin, on the synthesis, maturation and release of vesicular stomatitis virus (VSV) in cultures of Chinese hamster ovary (CHO) cells and the monensin-resistant clone, MonR-31. Our results depended on the dose and time of the addition of monensin to the infected cells, from 1 h prior to VSV infection to 1 h after infection. VSV production was more resistant in MonR-31 than in CHO cells when the ionophore was added 1 h prior to VSV infection. Monensin added 1 h after VSV infection showed the opposite phenomenon; release of virus particles into the medium was 10- to 10(5)-fold less in MonR-31 cells than in CHO cells, and the intracellular virus number in the resistant cells was one-third to one-fourth of that in the parental CHO cells. Syntheses of all virus-associated G, N and M proteins were inhibited in both cell lines by monensin, but especially so in the MonR-31 cells. There were no marked qualitative changes in the biochemical properties of viral glycoprotein G in virus-infected CHO and MonR-31 cells treated with monensin after virus infection. An endoglycosidase H-resistant G with a molecular weight smaller than that of normal G and attachments of palmitate or fucose on the truncated G protein appeared. Alteration of the secretion of as well as the synthesis of the enveloped virus is discussed in relation to the monensin susceptibility of the resistant MonR-31 clone.     

A fluorescence photobleaching study of vesicular stomatitis virus infected BHK cells. Modulation of G protein mobility by M protein

The mobility of vesicular stomatitis virus (VSV) G protein on the surface of infected BHK cells was studied by using the technique of fluorescence photobleaching recovery. The fraction of surface G protein that was mobile in that time scale of the measurement (minutes) was at least 75%, a relatively high value among cell surface proteins so far observed. For studies of the effect of an internal viral protein (M protein) on G protein mobility, cells infected with wild-type VSV were compared with those infected with temperature-sensitive VSV mutants of complementation group III, which contains lesions in the M protein. At the permissive temperature, a pronounced decrease in the mobile fraction of surface G was observed for each of three mutants studied, while mobility of surface G at the nonpermissive temperature was indistinguishable in mutant and wild-type infected cells. A significantly lower mobile fraction of G protein was also observed in SV40 transformed 3T3 cells infected with wild-type VSV, but not in 3T3 or chick embryo fibroblast cells similarly infected. None of the variables tested had a measurable effect on the lateral diffusion coefficient of the mobile G protein. These results are interpreted as modulation of the mobility of a specific cell surface protein by a specific intracellular protein.     

Inhibition of vesicular stomatitis virus replication in dexamethasone-treated L929 cells

We previously demonstrated that dexamethasone treatment of L929 cells inhibited plaque formation by vesicular stomatitis virus (VSV), encephalomyocarditis virus, or vaccinia virus. We now have characterized the antiviral effects of glucocorticoids in L929 cells. Dexamethasone did not directly inactivate VSV nor did steroid treatment of L929 cells affect virion adsorption or penetration. The VSV yield in L929 cells treated with dexamethasone for a period of only 4 or 8 hr was decreased by 50% when cells were infected the day following steroid treatment. Treating L929 cells with dexamethasone for a longer period resulted in greater inhibitions of virus synthesis. Interferon activity (less than 5 units/ml) was not detected in L929 cell culture fluids and cell sonicates from steroid-treated cells and the addition of antiserum to murine alpha/beta-interferon had no effect on the ability of dexamethasone to inhibit VSV replication. Dexamethasone treatment of L929 cells did not induce the production of double-stranded RNA-dependent protein kinase but did result in a slight elevation of 2-5A oligoadenylate synthetase activity, two enzymatic activities associated with the antiviral state induced by interferon. However, the elevated 2-5A synthetase activity was not associated with an inhibition of VSV RNA accumulation in dexamethasone-treated L929 cells. By contrast, the synthesis of all five VSV proteins was reduced by 50-75% in dexamethasone-treated L929 cells as early as 4 hr after infection. Thus, the dexamethasone-mediated inhibition of VSV replication in L929 cells is associated with decreased production of VSV structural proteins.