Human cell surface proteins selectively assembled into vesicular stomatitis virus virions

Vesicular stomatitis virus (VSV) selectively assembled proteins from human cells into progeny virions. These proteins can be surface labeled before infection with 125I, and when purified virus was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, only two or three bands of proteins (Mr around 100K) were seen. Antisera to these proteins were produced, using as immunizing antigen VSV tsO45 mutant, defective in assembly of G protein, which had been made at the nonpermissive temperature in the three human tumor cell lines, HeLa (cervical carcinoma), T47D (breast carcinoma), and HMB2 (melanoma). After absorption with wild-type VSV, each of the antisera displayed a different pattern of reactivity; at least three antigenic specificities were detected. Two of them, corresponding to antigens selected by VSV from HeLa and T47D, were to some extent related and they showed an association mainly with epithelial cell-derived gynecological tumors, but they were absent in carcinomas of lung or of digestive tract. These (or related) antigens were expressed in a lower level in some normal tissues, mainly in ovaries. Antigen(s) assembled by VSV from the melanoma cell line was entirely different and appeared to be associated with cell growth. The grounds for selective assembly of these specific proteins by VSV are not clear; they either share with viral surface glycoproteins some physical or structural properties, which are critical for incorporation into the viral envelope, or conceivably they even may represent uncleaved precursor proteins coded by env genes of incomplete genomes of endogenous human retroviruses.     

Frequent generation of new 3'-defective interfering particles of vesicular stomatitis virus

We have isolated and partially characterized a number of different genome types of defective interfering (DI) particles newly generated by a highly heat-resistant strain of vesicular stomatitis virus in either Rat(B77) or Vero cells. Northern blot analyses revealed that many of these DI genomes contain N gene sequences and/or sequences of the NS, M, and G genes. One type contains NS sequences without any indication for the presence of either N, M, or G sequences. Another type of DI particle genomes did not contain any detectable sequences of N, NS, M, or G, but contain panhandle-type sequences and, thus, most likely resembles the 5'-panhandle-type DI particles. Unlike previously assumed, these data demonstrate that DI genomes which have the 3'-terminal N, NS, M, and G genes or portions of these genes conserved do frequently arise together with 5'-DI particle genomes after serial undiluted passages of the heat-resistant strain of vesicular stomatitis virus.     

Reversible restriction of vesicular stomatitis virus in permissive cells treated with inhibitors of prostaglandin biosynthesis

Indomethacin, a potent nonsteroidal inhibitor of prostaglandin synthetase (cyclooxygenase) reduced yields of infectious vesicular stomatitis virus in HEp-2 cells more than 99% if added to cultures at levels of 10^?3M either before or after infection. Other permissive cell lines differed according to the treatment period and drug level required for restricting productive infections. The inhibitory effect of indomethacin was progressively reduced if infection of cells was delayed for increasing times after drug removal. Strong inhibition of viral replication also occurred in cells treated with the cyclooxygenase antagonists naproxen, phenylbutazone, and oxyphenylbutazone whereas phenacetin, which does not block cyclooxygenase function, was inactive. Enhanced viral replication occurred in indomethacin-treated HEp-2 cultures when these cells were subsequently exposed to such substances as prostaglandin El, cyclic AMP, or insulin. Conversely, indomethacin-treated cells remained restrictive for VSV if they were subsequently exposed to metabolic inhibitors of functional DNA (actinomycin D or mitomycin C), messenger RNA synthesis (α-amanitin), or protein synthesis (cycloheximide) at concentrations that normally do not compromise viral replication. Pretreatment of HEp-2 cells with mitomycin C markedly shifted the dose response for indomethacin-mediated inhibition of VSV from a 90% inhibitory dose of about 10^?4 M to one of 10^?9 M or lower. These findings suggest that preexisting host factors essential for replication of VSV, although rendered nonfunctional by the drug indomethacin, can be replenished unless their synthesis is blocked by various classes of metabolic inhibitors.     

Complete nucleotide sequence of the mRNA coding for the N protein of vesicular stomatitis virus (New Jersey serotype)

The nucleotide sequence of the mRNA encoding the nucleocapsid protein of the New Jersey serotype (Ogden strain) of vesicular stomatitis virus (VSV) was determined from two overlapping cDNA clones spanning almost entirely the coding region of the mRNA. The 5'-terminal noncoding sequence present in the mRNA but not in the cDNA clones was determined from a primer extended to the 5' terminus of the mRNA. The mRNA is 1329 nucleotides long (excluding polyadenylic acid) and encodes a protein of 422 amino acids. The nucleotide sequence was compared with the previously determined nucleotide sequence of the nucleocapsid protein of the Indiana serotype. An overall identity of 67.7% was found between the two serotypes. The only place where insertions and/or deletions have occurred during the evolution of the two viral genes from their presumed common ancestor is in the untranslated region. The nonidentical nucleotides are distributed throughout the length of the mRNA although not in an entirely random manner. The predicted amino acid sequence demonstrates that both proteins are initiated from the initiator codon located at the same distance from the 5' end (nucleotides 14 to 16) and contain the same number of amino acids. An overall identity of more than 80% of the amino acid sequence was observed between the two proteins when conservative replacements of amino acids were considered.     

Interactions of plus and minus strand leader RNAs of the New Jersey serotype of vesicular stomatitis virus with the cellular La protein

The New Jersey serotype of vesicular stomatitis virus (VSV-NJ) was found to synthesize a minus strand leader RNA of 44-46 bases long and a plus strand leader RNA of 47-50 bases long in infected cells. The minus strand leader RNA of VSV-NJ was found associated with the host cell La protein in infected cells by immunoprecipitation with antisera from patients with systemic lupus erythematosus. These results differ from those reported previously (J. Wilusz , M. G. Kurilla , and J. D. Keene (1983). Proc. Natl. Acad. Sci. USA 80, 5827-5831) for the similarly sized species of minus strand leader RNA made by the Indiana serotype of VSV (VSV-IND). Despite sequence differences between the 3' ends of the plus strand leader RNAs of the two serotypes, the plus strand leader RNA of VSV-NJ was found to have a pattern of La protein accumulation similar to that reported previously for the plus strand leader RNA of VSV-IND. These results provide additional support for a role for La protein in VSV replication and help further delineate the sequence requirements for La protein binding to VSV leader RNAs.     

Characterization of New Jersey vesicular stomatitis virus isolates from horses and black flies during the 1982 outbreak in Colorado

Vesicular stomatitis viruses isolated from horses, afflicted during the recent outbreak in the western United States, and from black flies (Simuliidae) were characterized with respect to the homology of their genomic RNAs and the mobility of their proteins in polyacrylamide gels. All the isolates were very similar, if not identical, with respect to these two parameters. When the black fly isolate was compared to other VSV isolates, this virus appeared to belong in the Hazelhurst subgroup of the New Jersey serotype of VSV. Since the other viruses in this division were obtained from infected swine, the natural host range of this subgroup has been extended to horses.     

Cross-linking of viral RNA by 4'-aminomethyl-4,5',8-trimethylpsoralen in HeLa cells infected with encephalomyocarditis virus and the tsG114 mutant of vesicular stomatitis virus

The presence of double-stranded RNA (dsRNA) of viral origin in infected cells is controversial. We established that in intact HeLa cells infected with encephalomyocarditis virus (EMCV) viral RNA can be cross-linked with 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT). This compound intercalates into dsRNA and forms cross-links upon irradiation with uv light. Addition of AMT to EMCV-infected cells, followed by irradiation, resulted in a dose-dependent inhibition of viral RNA synthesis, This inhibition was correlated with the cross-linking of nascent RNA strands to template strands of the viral replicative intermediate of EMCV. In contrast, treatment with AMT of vesicular stomatitis virus (VSV)-infected cells had no effect on viral mRNA synthesis and no cross-linking of viral RNA could be detected. The synthesis of viral RNA by the tsG114 mutant of VSV at the nonpermissive temperature, however, was inhibited by AMT. In cells infected with this mutant, cross-linked viral RNA could be detected. These results are discussed with regard to the role of dsRNA in cellular responses to viral infection mediated by interferon.     

Interaction of enveloped viruses with planar bilayer membranes: observations on Sendai, influenza, vesicular stomatitis, and Semliki Forest viruses

Exposure of a planar lipid bilayer to Sendai virus at pH 7.0 resulted in conductance increases that continued over several minutes, provided that the virus particles had first been conditioned by freezing and thawing, sonicating, or storing for 2 weeks in the cold. Individual electrical events could not be resolved, even on a millisecond time scale, and thus do not reflect the insertion of structural channels into the lipid bilayer. Prior treatment of the Sendai virions with protease prevented the conductance increases, but exposure of the bilayer to protease after induction of the conductance change did not abolish it. The Sendai-induced conductance change was increased in rate, but qualitatively unchanged in nature, if gangliosides were included in the planar bilayer. Activity for Sendai virus was low at pH 5.0, and increased with increasing pH up to 9.0. Influenza, Semliki Forest virus, and vesicular stomatitis virus all induced similar conductance changes around pH 5.2, but were inactive when tested at pH 7.0. The presence of cholesterol in the bilayer caused marked enhancement (two- to sixfold) of the response to Sendai, influenza and Semliki Forest virus, but caused only slight enhancement of the response to vesicular stomatitis virus. It is concluded that the observed increases in ionic permeability arise from alterations in lipid motions on a submillisecond time scale resulting from the incorporation of damaged viral membranes into the planar bilayer by fusion.     

Subcellular localization of the v-erb-B protein,the product of a transforming gene of avian erythroblastosis virus

Avian erythroblastosis virus (AEV) is an oncogenic retrovirus capable of transforming both fibroblasts and immature erythroid cells. The v-erb-B locus within the AEV genome encodes a glycosylated protein, expression of which is required for oncogenic transformation of either cell type. Subcellular localization of the v-erb-B glycoprotein in AEV-transformed cells is reported here. Results indicate that the v-erb-B protein is synthesized on dense membrane fractions and appears to possess the properties of an integral membrane protein. The bulk of the v-erb-B protein remains with dense membranes after synthesis, although a small quantity may slowly become associated with the plasma membrane. The biogenesis and subcellular location of the v-erb-B protein are thus quite different from those of the transforming proteins that display protein kinase activity. These differences are especially provocative because the amino acid sequences of the v-erb-B protein and the protein kinases are closely related to one another.     

Reversible block in intracellular transport and budding of mutant vesicular stomatitis virus glycoproteins

The blocks in intracellular maturation of glycoprotein (G protein) synthesized by two temperature-sensitive vesicular stomatite's virus (VSV) mutants are reversible. Our earlier work demonstrated that at 40 degrees, the nonpermissive temperature, mutant ts L513(V) G protein accumulates in the rough endoplasmic reticulum. Here we show that when the temperature is lowered the high-mannose oligosaccharides on a significant fraction of ts L513(V) G protein, synthesized at 40 degrees, will be modified to the complex type. Moreover, when the temperature is lowered ts L513(V) G protein accumulated at 40 degrees will mature to the cell surface, as evidenced by its accessibility to extracellular trinitrobenzene sulfonate, and into virions. G protein synthesized at 40 degrees in ts L511(V)-infected cells undergoes most of the processing events characteristic of the Golgi complex. Although we reported previously that no ts L511(V) G protein reaches the plasma membrane at 40 degrees, we now find, using more sensitive techniques, that an appreciable fraction does reach the cell surface. ts L511(V) G protein is lost from the cells but is not incorporated into virions. However, an appreciable fraction of the ts L511(V) G protein which accumulates in cells at 40 degrees will mature into virions when the temperature is lowered. These results exclude irreversible denaturation of mutant G proteins as a cause of the block in intracellular maturation and virus budding.