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.     

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.     

Isolation and replication of rabies virus in C6 rat glioma cells (clone CCL-107).

The susceptibility of the C6 rat glioma cell line (ATCC; CCL-107) to rabies virus was characterized. The kinetics of infection performed with a fixed and a wild strain (from an infected cow) of rabies virus was monitored by direct immunofluorescence. Fluorescent cytoplasmic bodies were readily observed by UV microscopy from 24 hours post-infection (hpi) onwards. The ability of C6 to produce rabies infective virion particles was confirmed by determining the viral titres present in the supernatants of infected cultures, by both BHK-21 cell infection and mice inoculation. C6 cells produced similar viral titres to those produced by BHK-21 for both strains used. In addition, the yield of rabies glycoprotein was assessed by ELISA. In general, BHK-21 and C6 cells infected either by PV or with the wild rabies strain produced similar amounts of rabies glycoprotein. At 96 hpi, however, when the glycoprotein production peaked, BHK-21 infected with the wild strain produced significantly higher amounts of glycoprotein than C6. Subsequently, the optimal conditions for isolation of wild rabies virus strains from C6 cells were established and these proved to be as sensitive as NA cells in detecting 10 wild rabies samples. Due to the high sensitivity exhibited, C6 rat glioma cells present a new and useful system for rabies virus investigation.     

Immune sera and antiglycoprotein monoclonal antibodies inhibit in vitro cell-to-cell spread of pathogenic rabies viruses.

Although the cell-to-cell spread of many viruses in vitro is inhibited by antibody, the effect of antibody on such spread of rabies viruses is uncertain. Thus, we examined the effects of anti-rabies virus immune sera and monoclonal antibodies (MAbs) on the in vitro spread of pathogenic rabies viruses in neuronal and nonneuronal cells. Both anti-rabies virus immune sera and neutralizing antiglycoprotein MAbs inhibited the cell-to-cell spread of street rabies virus, challenge virus standard, and ERA rabies viruses in cultures of neuroblastoma cells and of nonneuronal BHK-21 and chicken embryo-related cells. Furthermore, the cell-to-cell spread of virus was inhibited by greater than or equal to 75% with less than 1 IU/ml of human antirabies immunoglobulin. Nonneutralizing antinucleocapsid MAbs did not inhibit viral spread. After the immune serum was removed from the monolayers, virus spread rapidly to uninfected cells. Thus, antibody controlled the cell-to-cell spread of the virus but did not eliminate it from the cultures. Because antibody was more effective in inhibiting viral spread in fibroblast and epithelioid cells than in neuroblastoma cells infected at a high multiplicity of infection, we suggest that the inhibition of viral cell-to-cell spread by antibody in vivo would more likely occur at an initial site of exposure and before nerves are infected.     

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.     

Effect of certain inhibitors of glycoprotein synthesis on cell fusion induced by vesicular stomatitis virus.

The effect of certain metabolic inhibitors on the fusion of BHK-21 cells induced by vesicular stomatitis virus (VSV) was studied. The polykaryocyte formation in infected cells and virus growth were inhibited by 2-deoxy-D-glucose and D-glucosamine. Host-cell proteins synthesis was suppressed profoundly in both BHK-21-KB and B cells infected with VSV. On the other hand, glycoprotein synthesis was significantly enhanced during the polykaryocyte formation in BHK-21-KB cells, while it was suppressed in BHK-21-B cells which were not sensitive to cell fusion by VSV.     

Fusion properties of cells infected with human parainfluenza virus type 3: receptor requirements for viral spread and virus-mediated membrane fusion.

Cells can be persistently infected with human parainfluenza virus type 3 (HPF3) by using a high multiplicity of infection (MOI) (> or = 5 PFU per cell). The persistently infected cells exhibit no cytopathic effects and do not fuse with each other, yet they readily fuse with uninfected cells. We have previously shown that the failure of the persistently infected cells to fuse with each other is due to the lack of a receptor on these cells for the viral hemagglutinin-neuraminidase glycoprotein, and we have established that both fusion and hemagglutinin-neuraminidase proteins are needed for cell fusion mediated by HPF3. We then postulated that the generation of persistent infection and the failure of cells infected with HPF3 at high MOI to form syncytia are both due to the action of viral neuraminidase in the high-MOI inoculum. In this report, we describe experiments to test this hypothesis and further investigate the receptor requirements for HPF3 infection and cell fusion. A normally cytopathic low-MOI HPF3 infection can be converted into a noncytopathic infection by the addition of exogenous neuraminidase, either in the form of a purified enzyme or as UV-inactivated HPF3 virions. Evidence is presented that the receptor requirements for an HPF3 virus particle to infect a cell are different from those for fusion between cells. By treating infected cells in culture with various doses of neuraminidase, we demonstrate that virus spreads from cell to cell in the complete absence of cell-cell fusion. We compare the outcome of HPF3 infection in the presence of excess neuraminidase with that of another paramyxovirus (simian virus 5) and provide evidence that these two viruses differ in their receptor requirements for mediating fusion.     

Cell fusion activities of Hantaan virus envelope glycoproteins

Hantaan virus (HTNV)-infected Vero E6 cells undergo cell fusion with both infected and uninfected cells under low-pH conditions. Flow cytometry and fluorescence microscopy of HTNV-infected Vero E6 cells showed that envelope glycoproteins (GPs) were located both on the cell surface and in the cytoplasm. Neutralizing monoclonal antibodies (MAbs) against the G1 and G2 envelope GPs inhibited cell fusion, whereas nonneutralizing MAbs against G1 or G2 and MAbs against the nucleocapsid protein (NP) did not. Transfected Vero E6 cells that expressed GPs but not those that expressed NP fused and formed syncytia. These results indicate that HTNV GPs act as fusogens at the cell surface. No fusion activity was observed either in infected Vero cells that were passaged more than 150 times or in BHK-21 cells, although GPs appeared to localize to the cell surface. This variability in fusion induction suggests the involvement of host cell factors in the process of cell membrane fusion.     

Growth characteristics in cell culture and pathogenicity in mice of two terrestrial rabies strains indigenous to Canada

Two strains of street rabies virus from striped skunks (Mephitis mephitis) were used to infect either a murine neuroblastoma (NA 1300) or a baby hamster kidney (BHK-21/C13) cell culture and the cell infection rates were noted during 4 days postinfection. These cultures were then passaged for four consecutive passages, and the viruses obtained in the supernatant fluids of passage 4 were then treated as original isolates and used to infect both neuroblastoma and baby hamster kidney cells. The mortality period in Swiss white mice caused by the various virus suspensions was noted. The virus strain from the brain of skunks from Saskatchewan infected neuroblastoma and baby hamster kidney cells equally well, produced similar virus titres in supernatant fluids after four subcultures in both cell types, and appeared to produce similar mortality periods in mice from either the original brain tissue or from cell culture supernatant fluids. On the other hand, the virus from the brains of skunks from Ontario readily infected neuroblastoma but poorly infected baby hamster kidney cell cultures. Passage of this strain through four subcultures in both cell types produced virus titres in the supernatant fluids of equal magnitude. However, reisolation of the virus from the supernatant fluid of passage 4 in neuroblastoma cell cultures showed a similar pattern to that from the original brain, while the virus from baby hamster kidney cell passage supernatant fluid was considerably altered. Although the mortality period in mice was similar with virus from the brain and neuroblastoma cell cultures, this period was shortened when mice were inoculated with baby hamster kidney culture supernatant virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell surface expression of fusogenic vesicular stomatitis virus G protein from cloned cDNA.

Vesicular stomatitis virus (VSV) enters the host cell by the receptor-mediated endocytotic pathway. This brings the virus particle into acidic vesicles inside the cell where infection occurs through a fusion event between the viral and the host vesicle membrane. In this work we have shown that the VSV glycoprotein (G) carries the fusion activity of this virus. The G protein was expressed on the surface of baby hamster kidney 21 cells from cloned cDNA which had been engineered into an expression vector and introduced into cell nuclei with the aid of a glass microneedle. A short (60 s) treatment with acid (pH less than or equal to 6.0) medium induced fusion of cells having G protein on their surface. For efficient G protein expression and cell-cell fusion we had to trim the 5' end of the G cDNA and to use as promoter the long terminal repeat of the mouse Moloney sarcoma virus.     

Persistent infection of some standard cell lines by lymphocytic choriomeningitis virus: transmission of infection by an intracellular agent.

Cell-free cytoplasmic extracts of the Syrian hamster cell lines C13/SV28 and BHK-21F were immunogenic in Syrian hamsters. The resulting antisera cross-reacted completely with antisera against lymphocytic choriomeningitis virus (LCMV) in an immunoradiometric assay employing BHK-21F antigen. Several other Syrian hamster cell lines not previously known to be infected with LCMV were also strongly positive when assayed for viral antigens. Also, several mouse sera and antisera raised in Syrian hamsters against cells transformed by papovaviruses had high titers of anti-LCMV activity. No cytopathic effect was evident in any of the persistently infected cell lines. Culture media from these cells were not infectious and showed no evidence of defective interfering particles. However, cell-free extracts of all the persistently infected cells contained material capable of transmitting the persistent infection to uninfected cells of Syrian hamsters, rats, mice, green monkeys, and humans. The onset of infection is much slower than when LCMV virions are used. When 2 X 10(6) uninfected BHK cells were treated with an extract from 100 persistently infected cells, the new infection was apparent within about 12 days. When an extract from 10(6) cells was used, the new infection was apparent within about 5 days, but not sooner. The intracellular infectious material was sensitive to treatment with deoxycholate, Nonidet P-40, or ether but resistant to treatment with RNase or trypsin. It was also large (5,000S) and heterodisperse on sucrose gradients. The infectious material was probably contained in large lipid vesicles and their integrity was probably essential for infection. When a few persistently infected cells were cocultivated with many uninfected cells, a few discrete colonies positive for LCMV antigens were observed after about 5 days. Since the culture media were not infectious, the infection probably spread by cell-cell contact. Several different experiments indicated that interferon did not play a major role in mediating persistence in this case. Persistent infections by LCMV can be maintained without expression of extracellular virus particles and without appearance of large amounts of viral antigens on the cell surface. Cell-cell contact could still allow transmission of intracellular infectious material. In an animal, these properties could circumvent immune surveillance.     

Propagation of viruses on micropatterned host cells

We have developed a technique to characterize the in vitro propagation of viruses. Microcontact printing was used to generate linear arrays of alkanethiols on gold surfaces, which served as substrates for the patterned culture of baby hamster kidney (BHK-21) cells. Vesicular stomatitis virus (VSV) was added to unpatterned cell reservoirs adjacent to the patterned cells and incubated, setting in motion a continuously advancing viral infection into the patterned cells. At different incubation times, multiple arrays were chemically fixed to stop the viral propagation. Viral propagation distances into the patterned cells were determined by indirect immunofluorescent labeling and visualization of the VSV surface glycoprotein (G). The infection spread at approximately 50 microm/h in the 140-microm lines. Moreover, different temporal stages of the infection process were simultaneously visualized along individual lines. These stages included initiation of infection, based on G protein expression; cell-cell fusion, based on virus-induced clustering of cell nuclei; and cytoskeletal degradation, based on localized release of cells from the surface. This work sets a foundation for parallel, high-throughput characterization of viral and cellular processes.     

Growth of Rio Bravo Virus in Cell Cultures

The growth of Rio Bravo virus (RBV) in eight cell culture systems was studied. Highest yields of virus were produced in BHK-21 (C13), L, and Vero cell lines, but L cells were resistant to low doses of virus. LLC-MK(2), HeLa, and human embryo skin cells produced moderate amounts of virus, but FL amnion and primary chick embryo fibroblasts supported little virus growth. Virus was rapidly inactivated by exposure to pH values below 7.0. Single-cycle growth in BHK-21, L, and LLC-MK(2) cell monolayers was characterized by a latent period of about 12 hr followed by rapid virus production that peaked at 36 to 48 hr. Vero cell cultures can remain chronically infected with RBV for more than 100 days. Such cultures show evidence of cell destruction, and their supernatant fluids contain virus at 10(4) to 10(5) log(10) per ml.     

Antiapoptotic but Not Antiviral Function of Human bcl-2 Assists Establishment of Japanese Encephalitis Virus Persistence in Cultured Cells

Upon infection of Japanese encephalitis virus (JEV), baby hamster kidney (BHK-21) and Chinese hamster ovary (CHO) cells were killed by a mechanism involved in apoptosis. While readily established in a variety of cell lines, JEV persistence has never been successfully instituted in BHK-21 and CHO cells. Since stable expression of human bcl-2 in BHK-21 cells has been shown to delay JEV-induced apoptosis, in this study we investigated whether JEV persistence could be established in such cells. When constitutively expressing bcl-2, but not its closest homolog, bcl-X_L, following a primary lytic infection, approximately 5 to 10% of BHK-21 and CHO cells became persistently JEV infected during a long-term culture. From the persistent bulks, several independent clones were selected and expanded to form stable cell lines that continuously produced infectious virus without marked cytopathic effects (CPE). Among these stable cell lines, the truncated nonstructural protein 1 (NS1) was also detected and was indistinguishable from the NS1 truncations previously observed in JEV-persistent murine neuroblastoma N18 cells. However, the stable expression of NS1 alone, regardless of whether it was truncated or full length, failed to render the engineered cells persistently infected by JEV, implying that aberrant NS1 proteins were likely a consequence of, rather than a cause for, the viral persistence. Enforced bcl-2 expression, which did not affect virus replication and spread during the early phase of cytolytic infection, appeared to attain JEV persistence by restriction of virus-induced CPE. Our results suggest that it is the antiapoptotic, rather than the antiviral, effect of cellular bcl-2 which plays a role in the establishment of JEV persistence.     

Modification of Sindbis Virus Glycoprotein by Host-Specified Glycosyl Transferases

The amino acid sequence of the membrane glycoprotein of Sindbis virus is specified by the viral genome, but it has not been determined whether the carbohydrate portion of this molecule is specified by the cell or by the virus. We have examined two of the enzyme activities which catalyze transfer of monosaccharides to glycoprotein (sialyl and fucosyl transferases). Comparison of particulate enzyme preparations from infected and uninfected cells showed no difference in either the specific activity or acceptor specificity of these enzymes. This is impressive in view of the fact that the Sindbis membrane glycoprotein is the only glycoprotein synthesized in the infected cell. It was also determined that sialyl transferase from uninfected cells is capable of transferring ((3)H) sialic acid to acceptor prepared from Sindbis membrane glycoprotein. These results imply that at least some of the carbohydrate of the virus glycoprotein can arise by host modification.     

Effect of enforced expression of human bcl-2 on Japanese encephalitis virus-induced apoptosis in cultured cells.

Infection by Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes acute encephalitis in humans and induces severe cytopathic effects in different types of cultured cells. This study attempted to determine whether apoptosis contributes to virus-induced cell death in a culture system by characterizing JEV lytic infection in baby hamster kidney BHK-21 cells, murine neuroblastoma N18 cells, and human neuronal progenitor NT2 cells. According to our results, the replication of JEV, and not the UV-inactivated virions per se, triggered apoptosis in these cell lines, as evidenced by nuclear condensation, DNA fragmentation ladder, and in situ end labeling of DNA strand breaks with terminal transferase (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay). Different strains of JEV, regardless of whether they are neurovirulent to mice, could induce apoptosis of the infected cells. In addition, enforced expression of the human protooncogene bcl-2 in BHK-21 cells, which did not influence virus production, appeared to delay the process of JEV-induced apoptosis, despite the fact that most infected cells were inevitably killed after prolonged cultures. However, Bcl-2 proteins expressed in N18 cells failed to block JEV-induced apoptosis, although they did prevent Sindbis virus-induced apoptosis from occurring in the same cells. This finding suggests that these two viruses may utilize similar but not identical mechanisms to kill their infected cells. The results presented here thus demonstrate that apoptosis can be a general mechanism for JEV-induced cell death and that enforced bcl-2 expression may be inadequate in protecting all cell types from JEV-induced apoptosis in cell cultures.     

The apparent infection of NA-C1300 cell cultures by nucleocapsid material of the Canadian Arctic strain of rabies virus

Murine neuroblastoma (NA-C1300) and baby hamster kidney (BHK-21/C13) cell cultures were infected with the Canadian Arctic strain of rabies virus. Subcultures were passed following incubation for 3 to 4 days at 35 degrees C. The supernatant fluids from the BHK cultures demonstrated increasing infectivity in both NA and BHK cells concomitantly with an increase in the number of parent cells staining with an anti-glycoprotein stain. On the other hand, the supernatant fluids from the NA cultures initially showed higher infectivity in NA cells than in BHK cells. This feature was related to a low production of glycoprotein-staining cells in the parent NA cultures. The reduction of infectivity in NA cells of some NA supernatant fluids (and brain suspensions) by anti-nucleoprotein antibodies suggests that nucleocapsid material is, in some manner, capable of infecting NA cells. Infectivity of this virus strain in experimental mice is also related to the production of glycoprotein and may not be correlated with the degree of infection in NA cell cultures.     

Antibodies to CD9, a tetraspan transmembrane protein, inhibit canine distemper virus-induced cell-cell fusion but not virus-cell fusion

Canine distemper virus (CDV) causes a life-threatening disease in several carnivores including domestic dogs. Recently, we identified a molecule, CD9, a member of the tetraspan transmembrane protein family, which facilitates, and antibodies to which inhibit, the infection of tissue culture cells with CDV (strain Onderstepoort). Here we describe that an anti-CD9 monoclonal antibody (MAb K41) did not interfere with binding of CDV to cells and uptake of virus. In addition, in single-step growth experiments, MAb K41 did not induce differences in the levels of viral mRNA and proteins. However, the virus release of syncytium-forming strains of CDV, the virus-induced cell-cell fusion in lytically infected cultures, and the cell-cell fusion of uninfected with persistently CDV-infected HeLa cells were strongly inhibited by MAb K41. These data indicate that anti-CD9 antibodies selectively block virus-induced cell-cell fusion, whereas virus-cell fusion is not affected.     

Complement-Fixing Antigen from BHK-21 Cell Cultures Infected with Lymphocytic Choriomeningitis Virus

Infection of BHK-21 cells with lymphocytic choriomeningitis (LCM) virus resulted in the production of significant titers of complement-fixing (CF) antigen. The antigen was spontaneously released from the cells, but the highest titer of 1:16 was recovered by disruption of the infected cells by freeze-thawing in tryptose phosphate broth. The antigen could be partially separated from infectious virus by centrifugation. Furthermore, it was possible to detect LCM virus infection of cell cultures by the production of the CF antigen, but this method proved less sensitive than titration by intracerebral inoculation of mice. The CF antigen from cell cultures was at least as sensitive and specific as the reference antigen prepared from infected guinea pig spleen.