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.     

Viral glycoproteins destined for apical or basolateral plasma membrane domains traverse the same Golgi apparatus during their intracellular transport in doubly infected Madin-Darby canine kidney cells

Madin-Darby canine kidney (MDCK) cells can sustain double infection with pairs of viruses of opposite budding polarity (simian virus 5 [SV5] and vesicular stomatitis virus [VSV] or influenza and VSV), and we observed that in such cells the envelope glycoproteins of the two viruses are synthesized simultaneously and assembled into virions at their characteristic sites. Influenza and SV5 budded exclusively from the apical plasma membrane of the cells, while VSV emerged only from the basolateral surfaces. Immunoelectron microscopic examination of doubly infected MDCK cells showed that the influenza hemagglutinin (HA) and the VSV G glycoproteins traverse the same Golgi apparatus and even the same Golgi cisternae. This indicates that the pathways of the two proteins towards the plasma membrane do not diverge before passage through the Golgi apparatus and therefore that critical sorting steps must take place during or after passage of the glycoproteins through this organelle. After its passage through the Golgi, the HA accumulated primarily at the apical membrane, where influenza virion assembly occurred. A small fraction of HA did, however, appear on the lateral surface and was incorporated into the envelope of budding VSV virions. Although predominantly found on the basolateral surface, significant amounts of G protein were observed on the apical plasma membrane well before disruption of the tight junctions was detectable. Nevertheless, assembly of VSV virions was restricted to the basolateral domain and in doubly infected cells the G protein was only infrequently incorporated into the envelope of budding influenza virions. These observations indicate that the site of VSV budding is not determined exclusively by the presence of G polypeptides. Therefore, it is likely that, at least for VSV, other cellular or viral components are responsible for the selection of the appropriate budding domain.     

Antibody-induced modulation of proteins in vesicular stomatitis virus-infected fibroblasts.

When vesicular stomatitis virus-infected baby hamster kidney cells were treated with rabbit anti-vesicular stomatitis virus serum, there was a loss of the viral glycoprotein G into acid-soluble products. This degradation occurred within minutes at 37 degrees C and required the presence of G protein at the cell surface. The degree of degradation depended on antiserum concentration. The antiserum, also, prevented maturation of extracellular virions and induced partial degradation of the intracellular viral proteins, without affecting host proteins. The degradation could not be prevented by the presence of lysosomotropic agents, protease inhibitors, colchicine, or cytochalasin B. Similar kinetics and specificity of degradation was obtained with cells infected with vesicular stomatitis virus mutants that were less cytopathic. These results characterize a model system for studying the parameters and consequences of antigenic modulation as well as for studying the fate of viral antigens during persistent infections.     

Temperature-sensitive mutants of vesicular stomatitis virus are conditionally defective particles that interfere with and are rescued by wild-type virus.

Temperature-sensitive (ts) mutants of vesicular stomatitis virus belonging to complementation groups I, II and IV inhibited the replication of wild-type vesicular stomatitis virus when mixed infections were carried out in BHK21 cells at 32, 37, and 39.5 C. The group IV mutant (ts G 41) was most effective in this regard; wild-type virus yields were inhibited almost 1,000-fold in mixed infections with this mutant at 32 C. In the case of group I and II mutants, inhibition of wild-type virus replication at 37 and 39.5 C was accompanied by an enhancement (up to 15,000-fold) of the yields of the coinfecting ts mutant. The yields of the group IV mutant (ts G 41) were not enhanced by mixed infections with wild-type virus at any temperature, although this mutant inhibited wild-type virus replication at all temperatures. The dominance of the replication of ts mutants at 37 C provides a rationale for the selection and maintenance of ts virus in persistently infected cells.     

Basis for selective incorporation of glycoproteins into the influenza virus envelope.

The ability of mutant or chimeric A/Japan hemagglutinins (HAs) to compete for space in the envelope of A/WSN influenza viruses was investigated with monkey kidney fibroblasts that were infected with recombinant simian virus 40 vectors expressing the Japan proteins and superinfected with A/WSN influenza virus. Wild-type Japan HA assembled into virions as well as WSN HA did. Japan HA lacking its cytoplasmic sequences, HAtail-, was incorporated into influenza virions at half the efficiency of wild-type Japan HA. Chimeric HAs containing the 11 cytoplasmic amino acids of the herpes simplex virus type 1gC glycoprotein or the 29 cytoplasmic amino acids of the vesicular stomatitis virus G protein were incorporated into virions at less than 1% the efficiency of HAtail-. Thus, the cytoplasmic domain of HA was not required for the selection process; however, foreign cytoplasmic sequences, even short ones, were excluded. A chimeric HA having the gC transmembrane domain and the HA cytoplasmic domain (HgCH) was incorporated at 4% the efficiency of HAtail-. When expressed from simian virus 40 recombinants in this system, vesicular stomatitis virus G protein with or without (Gtail-) its cytoplasmic domain was essentially excluded from influenza virions. Taken together, these data indicate that the HA transmembrane domain is required for incorporation of HA into influenza virions. The slightly more efficient incorporation of HgCH than G or Gtail- could indicate that the region important for assembling HA into virions extends into part of the cytoplasmic domain.     

Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant.

We have used transient expression of the wild-type vesicular stomatitis virus (VSV) glycoprotein (G protein) from cloned cDNA to rescue a temperature-sensitive G protein mutant of VSV in cells at the nonpermissive temperature. Using cDNAs encoding G proteins with deletions in the normal 29-amino-acid cytoplasmic domain, we determined that the presence of either the membrane-proximal 9 amino acids or the membrane-distal 12 amino acids was sufficient for rescue of the temperature-sensitive mutant. G proteins with cytoplasmic domains derived from other cellular or viral G proteins did not rescue the mutant, nor did G proteins with one or three amino acids of the normal cytoplasmic domain. Rescue correlated directly with the ability of the G proteins to be incorporated into virus particles. This was shown by analysis of radiolabeled particles separated on sucrose gradients as well as by electron microscopy of rescued virus after immunogold labeling. Quantitation of surface expression showed that all of the mutated G proteins were expressed less efficiently on the cell surface than was wild-type G protein. However, we were able to correct for differences in rescue efficiency resulting from differences in the level of surface expression by reducing wild-type G protein expression to levels equivalent to those observed for the mutated G proteins. Our results provide evidence that at least a portion of the cytoplasmic domain is required for efficient assembly of the VSV G protein into virions during virus budding.     

Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus.

Maturation of viral proteins in cells infected with mutants of vesicular stomatitis virus was studied by surface iodination and cell fractionation. The movement of G, M, and N proteins to the virion bud appeared to be interdependent. Mutations thought to be in G protein prevented its migration to the cell surface, allowed neither M nor N protein to become membrane bound, and blocked formation of viral particles. Mutant G protein appeared not to leave the endoplasmic reticulum at the nonpermissive temperature, but this defect was partially reversible. In cells infected with mutants that caused N protein to be degraded rapidly or prevented its assembly into nucleocapsids, M protein did not bind to membranes and G protein matured to the cell surface, but never entered structures with the density of virions. Mutations causing M protein to be degraded prevented virion formation, and G protein behaved as in cells infected by mutants in N protein. These results are consistent with a model of virion formation involving coalescence of soluble nucleocapsid and soluble M protein with G protein already in the plasma membrane.     

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.     

Protein Kinase and Phosphoproteins of Vesicular Stomatitis Virus

Protein kinases of similar but not identical activity were found associated with vesicular stomatitis (VS) virions grown in mouse L cells, primary chicken embryo (CE) cells, and BHK-21 cells, as well as being present in VS virions grown in HeLa and Aedes albopictus cells. The virion kinase preferentially phosphorylated the nucleocapsid NS protein in vitro and to a lesser extent the envelope M protein. Other virion proteins were phosphorylated in vitro only after drastic detergent treatment. Partial evidence that the virion kinase is of cellular origin was obtained by finding reduced enzyme activity in virions released from cells pretreated with actinomycin D and cycloheximide. Selective detergent and detergent-salt fractionation of VS virions revealed that the kinase activity was present in the envelope but not the spikes. The virion kinase activity in a Triton-salt-solubilized envelope fraction could be separated from M and G proteins and partially purified by phosphocellulose column chromatography. Virions released from L, CE, and BHK-21 cells infected in the presence of [(32)P]orthophosphate were labeled almost exclusively in the NS protein. Both soluble and nucleocapsid-associated NS phosphoprotein were present in cytoplasmic extracts of VS viral-infected L cells. The origin and function of the NS phosphoprotein remain to be elucidated.     

Virus-Specific Proteins Synthesized in Cells Infected with RNA+ Temperature-Sensitive Mutants of Sindbis Virus

All Sindbis virus temperature-sensitive mutants defective in "late" functions were systematically surveyed by acrylamide-gel electrophoresis for similarities and differences in the intracellular pattern of virus-specific proteins synthesized at the permissive and nonpermissive temperatures. Only cells infected with mutants of complementation group C showed an altered pattern. At the nonpermissive temperature, these mutants failed to induce the synthesis of a polypeptide corresponding to the nucleocapsid protein and instead overproduced a protein of higher molecular weight than either viral structural protein. This defect was shown to be irreversible by the finding that (3)H-leucine incorporated at 41.5 C specifically failed to appear in the nucleocapsid of virions subsequently released at 29 C. Attempts to demonstrate a precursor protein in wild-type infections were inconclusive.     

Mutants deleted in the agnogene of simian virus 40 define a new complementation group

Analysis of the DNA sequence of the late leader region of simian virus 40 indicates that it might encode a 61-amino acid, highly basic protein, LP-1. Mutants deleted in this region are viable, but they produce infectious progeny more slowly than wild-type virus in established monkey cells. On the basis of the rates of appearance and the sizes of mixed plaques formed after cotransfections with pairs of mutants, we found that mutants defective in the synthesis of LP-1 complementation was also observed in infections with virions and was bidirectional. Therefore, these mutants define a new complementation group, group G. In addition, a protein of the appropriate molecular weight for LP-1 (approximately 8 X 10(3) ) was synthesized by wild-type virus-infected cells but not by mock-infected or group G gene mutant-infected cells. This protein, whose identity has been established definitively by Jay et al. (Nature (London) 291:346-349, 1981), was synthesized at a high rate at late times after infection, was present predominantly in the cytoplasmic fraction of cells, possessed a fairly short half-life, and was absent from mature virions. Once formed, virions of group G gene mutants behaved biologically and physically like virions of wild-type virus. On the basis of these findings and other known properties of LP-1 and mutants defective in LP-1 synthesis, we hypothesize that LP-1 functions to facilitate virion assembly, possibly by serving as a nonreusable scaffolding protein.     

Envelopment of Sindbis Virus: Synthesis and Organization of Proteins in Cells Infected with Wild Type and Maturation-Defective Mutants

The synthesis and organization of Sindbis virus structural proteins was investigated in BHK cells infected with wild-type virus (SVHR) or temperature-sensitive (ts) mutants defective in maturation. Cells infected with ts-23 or ts-20 (complementation groups D and E) were similar in the polypeptides synthesized at the nonpermissive temperature and differed from SVHR-infected cells in that the envelope protein E2 was not cleaved from the PE2 precursor. Data from experiments utilizing pulse-chase procedures or protein synthesis inhibitors indicated that although infectious virions were released from cells infected with these mutants in shift-down experiments, the particles were produced almost exclusively from proteins synthesized after the return to permissive temperature. This suggests that a stable complex may be formed among the structural proteins before budding. A membrane fraction isolated from cells infected with either ts mutants or SVHR contained the PE2, E1, and C polypeptides, whereas E2 was restricted to fractions obtained from SVHR-infected cells. Although equivalent amounts of virus-specific protein were synthesized in cells infected with either mutant and the cells contained qualitatively the same proteins in the isolated membranes, cells infected with ts-23 did not have virus-specific proteins exposed on their surface that could be detected by ferritin-conjugated antibody-labeling procedures or lactoperoxidase-mediated iodination. In contrast, ts-20-infected cells had significant amounts of viral protein, mainly E1, that could be detected on the plasma membrane by either procedure. Iodine was incorporated into E1 and E2 on the surface of SVHR-infected cells in the same relative amounts as seen in iodinated virions. PE2, however, although present in membranes, could not be iodinated on the surface of infected cells under any of the conditions used in this study. We also monitored the relative efficiency with which these viral proteins could be removed from intact cells by dilute solutions of nonionic detergents. The results indicated that E2 was most efficiently removed, followed by E1. PE2 (the precursor to E2) and C remained associated with the cell and could be subsequently isolated in the membrane fraction.