Requirement of carbohydrates for cell fusion induced by vesicular stomatitis virus

The fusion of BHK-21-KB cells by vesicular stomatitis virus was not induced in Eagle's minimal essential medium without glucose. In medium containing glucose, the rate of polykaryocyte formation decreased as the concentration of glucose was reduced below 5 mM. However, no reduction in virus production 24 hr after infection was seen under this condition. Addition of pyruvate or mannose to the culture medium caused a reversal of cell fusing activity. Cell fusion and virus growth were significantly suppressed by sodium azide and 2,4-dinitrophenol.     

Neuroblastoma Cell Fusion by a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus

A temperature sensitive mutant of vesicular stomatitis virus which does not mature properly when grown at 39 degrees C promoted extensive fusion of murine neuroblastoma cells at this nonpermissive temperature. Polykaryocytes apparently formed as a result of fusion from within the cells that requires low doses of infectious virions for its promotion and is dependent on viral protein synthesis. Although 90% of infected N-18 neuroblastoma cells were fused by 15 h after infection, larger polykaryocytes continued to form, leading to an average of 28 nuclei per polykaryocyte as a result of polykaryocytes fusing to each other. Two neuroblastoma cell lines have been observed to undergo fusion, whereas three other cell lines (BHK-21, CHO, and 3T3) were incapable of forming polykaryocytes, suggesting that nervous system-derived cells are particularly susceptible to vesicular stomatitis virus-induced fusion. Although the normal assembly of the protein components of this virus is deficient at 39 degrees C, the G glycoprotein was inserted into the infected cell membranes at this temperature. Two lines of evidence suggest that the expression of G at the cell surface promotes this polykaryocyte formation: (i) inhibition of glycosylation, which may be involved in the migration of the G protein to the cellular plasma membranes, will inhibit the cell fusion reaction; (ii) addition of antiserum, directed toward the purified G glycoprotein, will also inhibit cell fusion.     

Antibody-resistant spread of vesicular stomatitis virus infection in cell lines of epithelial origin.

In MDCK cells, vesicular stomatitis virus (VSV) buds exclusively from the basolateral plasma membranes beneath tight junctions, whereas influenza virus forms only at the free apical surface. Anti-VSV antiserum did not prevent the formation of plaques on MDCK cell monolayers infected with VSV, whereas plaque formation in BHK-21 cells was completely inhibited by such antiserum. Under similar conditions, homologous antiserum completely prevented plaque formation by influenza virus on MDCK cells. In several other epithelioid cell lines, VSV also formed plaques in the presence of specific antiserum. These results suggest that VSV receptors are present on basolateral membranes in the cells studied and that junctional complexes present between cells may exclude antibody from intercellular spaces and thus permit the lateral spread of virus infection in the presence of neutralizing antibody.     

Neuroblastoma Cell Membranes: Specificity for Cell Fusion Mediated by a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus

Abstract: Temperature-sensitive mutant G3 1 of vesicular stomatitis virus induces mouse neuroblastoma N-18 cells to fuse during infections that are nonpermissive for virus replication, but BHK-21 cells do not undergo the viral glycoprotein-mediated cell fusion. The viral glycoprotein was expressed at the cell surface of both N-18 and BHK-21 cells; therefore, the host cell specificity did not stem from an absence of the viral glycoprotein at the surface of BHK-21 cells. Cell fusion readily occurred between infected and uninfected N-18 cells in mixed cultures, demonstrating that the viral glycoprotein was interacting with an uninfected cell for the initial cell-cell interaction of the cell fusion. Mixing infected BHK-21 cells with uninfected N-18 cells resulted in cell fusion initiated by BHK-21 cell-synthesized viral glycoprotein, but 88% of the nucleiin polykaryocytes were N-18 nuclei. The N-18 cell fusion specificity was readily apparent when infected N-18 cells were mixed with uninfected BHK-21 cells; 98% of the nuclei in polykaryocytes were N-18 nuclei. Similar results also were obtained with mixed cultures of N-18 cells and primary astroglial cells. Thus, the viral glycoprotein synthesized in any of the cell types could initiate cell fusion, but the properties of plasma membranes of neuroblastoma cells appeared to be much more suitable for cell-cell fusion.     

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.     

Tumorigenicity of persistently infected tumors in nude mice is a function of both virus and host cell type.

Although BHK-21 cells persistently infected with wild-type vesicular stomatitis virus (VSV) are sensitive to natural killer (NK) cells and do not form tumors in athymic nude mice, BHK-21 cells persistently infected with a previously isolated mutant virus (VSV-P) are resistant to NK cells and form tumors in nude mice. We used this VSV-P mutant to persistently infect HeLa cells and mouse tumor cell lines. A mouse mastocytoma line (P815) persistently infected with VSV-P was similar to BHK-21 cells in that it was resistant to NK cell lysis and formed tumors in nude mice. However, neither HeLa cells nor mouse myeloma lines persistently infected with VSV-P were resistant to NK cell lysis in vitro, and neither formed tumors in nude mice. Rejection by nude mice of HeLa cells and mouse myeloma cell lines persistently infected with VSV-P could be ablated by rabbit antiserum to asialo-GM1, implicating NK cells in the in vivo rejection of these persistently infected tumors. These results suggest that NK cell recognition and killing of virus-infected cells in vivo and in vitro depend upon genetic contributions from both the virus and the host cell.     

Activation of mouse lymphocytes by vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) is a mitogen for mouse spleen cells, and infectious virus is not required for mitogenesis. At concentrations between 10 and 100 microgram per culture, VSV stimulated DNA synthesis and blast transformation. Maximal activation by VSV occurred 48 h after culture initiation. Spleen cells depleted of T-lymphocytes by treatment with anti-Thy 1.2 and complement and those obtained from congenitally athymic BALB/c nu/nu mice were activated by VSV, suggesting that VSV is a B-cell mitogen. Activation of spleen cells was independent of the host in which the virus was grown, since VSV grown in BHK-21, HKCC, or MDBK cells was mitogenic. The mitogenesis was specific for VSV, since MDBK cell-grown WSN influenza virus was not a mitogen in this in vitro activation system, VSV-specific antibody prevented VSV mitogenesis, and VSV was mitogenic for spleen cells from C3H/HeJ mice which were resistant to mitogenesis by endotoxin.     

Examination of Virus-Infected Cultured Cells by Scanning Electron Microscopy

The scanning electron microscope (SEM) was used to detect changes in morphology of BSC-1 cells after infection with vesicular stomatitis virus (VSV) or herpes simplex virus. The morphological changes of the infected cells were related to the length of time of infection and to the virus used. Extensive alteration to the cytoplasm could be seen 24 and 48 hr after infection with 10 and 320 TCID(50) of VSV. Within 24 hr after infection with 1 TCID(50) of herpes simplex, a few nuclei were swollen. However, 72 hr after infection with 100 TCID(50) of herpesvirus, many nuclei were swollen and appeared in large aggregates, probably representing formation of a polykaryocyte. Corresponding samples stained with May-Grunwald-Giemsa were observed in the light microscope and morphological changes were compared to those seen with the SEM.     

Vesicular stomatitis virus-mediated cell fusion subsequent to virus adsorption at different pH values.

Vesicular stomatitis virus (VSV)-mediated cell fusion from without can be induced by transient exposure to low pH, subsequent to adsorption of VSV at neutral pH. To study the mechanism of VSV-induced cell fusion, we examined the effect of pH condition at virus adsorption on acid-inducible VSV-mediated cell fusion. Although the binding of VSV to BHK-21 cells was most efficient under acidic condition (pH 5.7-6.3), extensive cell fusion was not observed under this condition. A temporary exposure to low pH after binding at neutral pH also decreased fusion activity. However, return to neutral pH for 2 min just after the acid binding restored the fusion activity. These results indicate the requirement of neutral pH condition for VSV-mediated cell fusion prior to the acid stimulation which induces conformational change of the virus glycoprotein into a fusogenic form.     

Glycopeptides from brain inhibit rates of polypeptide chain elongation

In previous reports, we have identified cell-surface glycopeptides from mouse cerebrum (BCSG) that inhibited protein synthesis and mitosis in several cell types. When baby hamster kidney (BHK)-21 cells were infected with vesicular stomatitis virus (a negative strand RNA virus), BCSG extensively inhibited viral protein synthesis. This inhibition was effective against both protein and glycoprotein synthesis and was independent of amino acid uptake by infected cells, synthesis of viral RNA, and degradation of viral proteins. Analysis of polyribosome profiles in uninfected BHK-21 cells indicated that the degree of cellular protein synthesis inhibition could not be attributed to activation of RNase or solely to a disruption of chain initiation. When added directly to a cell-free protein-synthesizing system derived from BHK-21 cells, BCSG was ineffective, but if the inhibitory material was first allowed to react with cells, cell-free protein synthesis was substantially reduced. When BCSG were reacted with cells for 5 min at 0 degrees C, the cells tested, BHK-21 (a BCSG-sensitive line) and murine fibrosarcoma 2237 (a BCSG-insensitive line), both effectively adsorbed the inhibitor from the medium.     

Failure of multinucleated giant cell formation in k562 cells infected with newcastle disease virus and human parainfluenza type 2 virus

When K562 cells were infected with Newcastle disease virus (NDV) or human parainfluenza type 2 virus (hPIV-2), polykaryocyte formation could not be detected. Failure of multinucleated giant cell formation in K562 cells infected with either NDV or hPIV-2 is due to disturbance of the viral envelope-cell fusion step or to defect in the cell-cell fusion step, respectively. Especially, NDV completely replicated in K562 cells, and the hemagglutinin-neuraminidase and fusion proteins expressed on the cell surface of NDV-infected K562 cell were fully functional for fusion inducing activity. Therefore, the cell membranes of K562 cells are considered to be resistant to virus-induced cell fusion. Membrane fusion is regulated by many host factors including membrane fluidity, cytoskeletal systems, and fusion regulatory proteins system. An unknown regulatory mechanism of virus-induced cell fusion may function on the cell surface of K562 cells.     

Growth and Maturation of a Vesicular Stomatitis Virus Temperature-Sensitive Mutant and Its Central Nervous System Isolate

A temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV), tsG31, produces a prolonged central nervous system disease in mice with pathological features similar to those of slow viral diseases. tsG31 and the subsequent virus recovered from the central nervous system (tsG31BP) of mice infected with tsG31 were compared with the parental wild-type (WT) VSV for plaque morphology, growth kinetics, thermal sensitivity of the virions, and viral protein synthesis and maturation. Several properties of the central nervous system isolate distinguished this virus from the original tsG31 and the WT VSV. The WT VSV produced clear plaques with complete cell lysis, and the tsG31 produced diffuse plaques and incomplete cell lysis, whereas the tsG31BP had clear plaques similar to those of the WT VSV. Although plaque morphology suggested that tsG31BP virus was a revertant to the WT, growth kinetics in either BHK-21 or neuroblastoma (N-18) cells indicated that this virus was similar to tsG31, with a productive cycle at 31 degrees C and no infectious virus at 39 degrees C. At 37 degrees C, however, the tsG31BP matured much slower than did the original tsG31 (and produced only 1% of the yield measured at 31 degrees C). WT VSV produced similar quantities of infectious virions at 31, 37, and 39 degrees C. The lack of infectious virions at 39 degrees C for the ts mutants was presumably not due to a greater rate of inactivation at 39 degrees C. Unlike WT VSV, which synthesized viral proteins equally well at all three temperatures, tsG31 had a reduced synthesis of all the structural proteins at 37 and 39 degrees C, compared with that at 31 degrees C; the formation of the M protein was most temperature sensitive. In addition, fractionation of the infected cells indicated that the incorporation of the M and N proteins into the cellular membranes was also disrupted at the higher, nonpermissive temperatures. Several characteristics of protein synthesis during tsG31BP infection at 39 degrees C distinguished this virus from tsG31: (i) no mature viral proteins were detected at 39 degrees C; (ii) several host proteins were [ill], suggesting that the virus was incapable of completely depressing host macromolecular synthesis; and (iii) a great proportion of the incorporated radioactivity was found in unusually high-molecular-weight proteins. In addition, at 37 degrees C, the tsG31BP virus showed a decreased synthesis of viral proteins and reduced assembly of the viral structural proteins.     

Inhibition of pseudorabies virus replication by vesicular stomatitis virus. II Activity of defective interfering particles.

Purified defective interfering (DI) particles of vesicular stomatitis virus (VSV) inhibit the replication of a heterologous virus, pseudorabies virus (PSR), in hamster (BHK-21) and rabbit (RC-60) cell lines. In contrast to infectious B particles of VSV, UV irradiation of DI particles does not reduce their ability to inhibit PSR replication. However, UV irradiation progressively reduces the ability of DI particles to cause homologous interference with B particle replication. Pretreatment with interferon does not affect the ability of DI particles to inhibit PSR replication in a rabbit cell line (RC-60) in which RNA, but not DNA, viruses are sensitive to the action of interferon. Under similar conditions of interferon pretreatment, the inhibition of PSR by B particles is blocked. These data suggest that de novo VSV RNA or protein synthesis is not required for the inhibition of PSR replication by DI particles. DI particles that inhibit PSR replication also inhibit host RNA and protein synthesis in BHK-21 and RC-60 cells. Based on the results described and data in the literature, it is proposed that the same component of VSV B and DI particles is responsible for most, if not all, of the inhibitory activities of VSV, except homologous interference.     

Proteins induced in tissue culture by four isolates of Theiler''s murine encephalomyelitis virus.

The proteins specified by four Theiler's murine encephalomyelitis virus isolates in infected BHK-21 cells were studied. Their processing, sensitivity to trypsin, and the changeover after viral infection from synthesis of cellular proteins to synthesis of viral proteins were determined by one- and two-dimensional gel electrophoreses. The molecular weights and isoelectric points of the structural and nonstructural proteins of DA and WW isolates, which represent the less virulent subgroup of Theiler's murine encephalomyelitis virus, and of GDVII and FA isolates, which represent the virulent subgroup, were found to be the same. The sensitivity of DA and GDVII isolates to trypsin, as purified virions, and in infected cell extracts was similar. The shut-off of cellular protein synthesis in cells infected with the same two isolates and the changeover to the synthesis of viral proteins appeared to have the same pattern. These findings are interesting since the two subgroups of Theiler's murine encephalomyelitis virus differ in their pathogenicity, intracellular development in infected BHK-21 cells, and RNA composition, as determined by RNase T1 fingerprinting analysis.     

Preliminary biochemical characterization of the factors(s) responsible for herpesvirus-induced exogenous fusion.

Cell-free extracts prepared from herpes simplex virus-infected BHK-21 cells rapidly induced exogenous fusion when incubated with indicator monolayers of uninfected BHK-21 cells. Fusion was first observed at 1 h, and peak activity was reached by 4 h. Divalent cations were required for activity. Inhibition of indicator cell macromolecular synthesis, with metabolic inhibitors, failed to prevent formation of cell-free extract-induced polykaryocytes. Removal of virus particles from the cell-free extract by velocity sedimentation centrifugation did not affect cell-free extract exogenous fusion activity. Studies using molecular probes, namely, glycosidases, lectins, and antiserum (directed against either HSV envelope or capsid proteins), suggest that the factor(s) responsible for herpesvirus fusion is a fucosylated glycoprotein that is not a structural component of the virion.     

Chemical cross-linking of proteins of Semliki Forest virus: virus particles and plasma membranes from BHK-21 cells treated with colchicine or dibucaine.

Chemical cross-linking of the proteins of Semliki Forest virus has been performed in virus particles and in baby hamster kidney-21 (BHK-21) cells infected with Semliki Forest virus. Most of the studies were done with the reversible cross-linkers dimethyl 3,3'-thiobis(propionimidate) and dithiobis(succinimidyl propionate). The identity of the cross-linked species was determined by two-dimensional electrophoresis. The results with virus particles showed extensive cross-linking of the nucleocapsid proteins and the formation of dimers of the two large envelope glycoproteins (E1 and E2). Similar patterns for the cross-linked virus proteins were observed in plasma membranes isolated from BHK-21 cells infected with Semliki Forest virus. No cross-linking of the third envelope glycoprotein (E3) was observed. Also, there was no evidence for significant cross-linking between host and virus proteins. The addition of colchicine, a drug that disrupts microtubules, to infected BHK-21 cells had no effect on the cross-linking of virus proteins in the plasma membrane. In contrast, dibucaine, a local anesthetic, greatly inhibited the formation of envelope dimers (E1-E2) in plasma membranes, but not in virus particles. The implication of these results for the involvement of the cytoskeletal system in the morphogenesis of Semliki Forest virus is discussed.     

Characteristics of Murayama virus in various cell cultures and laboratory animals

Some biological properties of Murayama virus, a new paramyxovirus, were studied. The virus grew well in primary monkey kidney cells as well as embryonated eggs, while the virus yields in primary chick embryo and BHK-21 cells were much lower. The infected BHK-21 cells formed large syncytia and showed typical hemadsorption, but did not produce any detectable amount of hemagglutinin in the culture fluid. The virus yields were very low in Vero. LLC-MK2 and MDCK cells at first passages. The addition of trypsin to the medium enhanced virus growth in Vero and LLC-MK2 but not in MDCK cells. Cell fusion activity of the virus was observed in Molt-4 cells. Hemolytic activity was enhanced by freeze-thawing. Several species of mammals and birds were susceptible to experimental infections with the virus as evidenced by seroconversion and positive virus isolation without showing any clinical signs.     

Lack of evolutionary stasis during alternating replication of an arbovirus in insect and mammalian cells

The evolution of vesicular stomatitis virus (VSV) in a constant environment, consisting of either mammalian or insect cells, has been compared to the evolution of the same viral population in changing environments consisting in alternating passages in mammalian and insect cells. Fitness increases were observed in all cases. An initial fitness loss of VSV passaged in insect cells was noted when fitness was measured in BHK-21 cells, but this effect could be attributed to a difference of temperature during VSV replication at 37 degrees C in BHK-21 cells. Sequencing of nucleotides 1-4717 at the 3' end of the VSV genome (N, P, M and G genes) showed that at passage 80 the number of mutations accumulated during alternated passages (seven mutations) is similar or larger than that observed in populations evolving in a constant environment (two to four mutations). Our results indicate that insect and mammalian cells can constitute similar environments for viral replication. Thus, the slow rates of evolution observed in natural populations of arboviruses are not necessarily due to the need for the virus to compromise between adaptation to both arthropod and vertebrate cell types.     

The entry into host cells of Sindbis virus, vesicular stomatitis virus and Sendai virus.

We have compared the mechanisms of entry into host cells of three enveloped viruses: Sendai virus, vesicular stomatitis virus (VSV) and Sindbis virus. Virus entry by membrane fusion should antigenically modify the surface of a newly infected cell in such a way that it will be killed by anti-viral antibody and complement. On the other hand, virus entry by a mechanism involving uptake by the cell of the whole virion should not make cells sensitive to antibody and complement. As expected, cells newly infected with Sendai virus were readily and completely lysed by anti-Sendai antibody and complement. In marked contrast, however, cells newly infected with either Sindbis virus or VSV were killed by anti-viral antibody and complement only when infected at an extremely high multiplicity of infection, in excess of 1000 plaque-forming units per cell. We favor the following explanation for these results with Sindbis virus and VSV: a very large majority of the Sindbis and VSV virions entered the infected cells by some means other than membrane fusion, presumably engulfment of the whole particle. Efficient entry by way of membrane fusion may therefore not be a general characteristic of enveloped viruses.     

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.