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.     

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.     

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.     

Ultrastructural localization of L and NS enzyme subunits on vesicular stomatitis virus RNPs using gold sphere-staphylococcal protein A-monospecific IgG conjugates

Colloidal gold spheres were coated with staphylococcal protein A and were used to determine the location of NS and L proteins on vesicular stomatitis virus (VSV) ribonucleoprotein (RNP) complexes using monospecific anti-NS and anti-L IgG preparations. Conjugates using either anti-NS or anti-L demonstrated that these enzyme subunits were uniformly distributed along the entire length of the RNP complex. Under saturating conditions of IgG concentrations, it was observed that there were at least 60-70 molecules of NS protein and 30-35 molecules of L protein labeled per RNP complex.     

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.     

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.     

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 west nile virus persistent infections in genetically resistant and susceptible mouse cells. II. Generation of temperature-sensitive mutants

Long-term persistent infections were established with the flavivirus, West Nile virus (WNV), strain E101, in embryofibroblast cultures derived from susceptible C3H/HE and congenic-resistant C3H/RV mice. Cultures were initially maintained by weekly subculture at 37 degrees, but at passage 6 sister cultures were shifted to 32 degrees. Virus progeny titers were observed to increase after the shift to 32 degrees indicating the possible presence of temperature-sensitive mutants. Temperature-sensitive mutants were found to arise in cultures of both susceptible and resistant cells. However, only in the resistant cultures did temperature-sensitive virus become the majority population. Temperature-sensitive mutants did not appear to be essential for either initiation or maintenance of WNV-persistant infections. The resistant cells appear to provide an environment which is advantageous for the amplification of temperature-sensitive mutants.     

Analysis of the terminal sequences of the genome segments of four orbiviruses

The dsRNA genome segments of bluetongue virus (BTV) types 1 and 20 and Ibaraki virus (a member of the epizootic haemorrhagic disease (EHD) serogroup) have conserved sequences of six bases at both of their 3' termini. One strand of all the genome segments analysed ends in 3'CAUUCA ... 5' while the other strand ends in 3'CAAUUU ... 5'. These conserved sequences are identical to those previously reported for BTV types 10 and 11 (A. Kiuchi, C. D. Rao, and P. Roy (1983), "Double-Stranded RNA Viruses" (R. W. Compans and D. H. L. Bishop, eds.), pp. 55-64. Elsevier, New York; C. D. Rao, A. Kiuchi, and P. Roy (1983), J. Virol. 46, 378-383). The 3' terminal sequences of segments 3 and 10 of the BTV type 1 genome were confirmed by the detection of exactly complementary sequences at the 5' termini of the ssRNA strands of opposite polarity. This also confirmed for these dsRNA segments (and by analogy for all the genome segments of these viruses) that the dsRNA molecules are fully base paired end to end. Using in vitro synthesised mRNA of BTV type 1 in annealing experiments with the two ssRNAs separated from each of the individual genome segments, it was shown that in each case the strand ending in 3'CAUUCA ... 5' is of the same polarity as the mRNA (+ve), while the strand ending in 3'CAAUUU ... 5' is of the opposite (-ve) polarity. The fourth virus analysed (Tilligerry virus, a member of the Eubenangee serogroup) only had five conserved bases at the 3' termini of one strand of its genome segments (3'CAU-CA ... 5') and three conserved bases at the 3' termini of the other strand (3'CA--U ... 5'). Considerable sequence homology was found in the near-terminal nonconserved regions of comparable genome segments from the different viruses, particularly between the different BTV types. There was little evidence, however, for absolute conservation of "segment specific" sequences in these regions of the RNA.     

Mapping of Epstein-Barr virus proteins on the genome by translation of hybrid-selected RNA from induced P3HR1 cells and induced Raji cells

RNA was isolated from induced P3HR1 cells which synthesize Epstein-Barr virus (EBV) particles and therefore a full set of early and late antigens and from induced Raji cells which synthesize only early EBV proteins and hybridized to cloned EBV-DNA fragments spanning the entire genome. Bound mRNA was eluted and translated in vitro with rabbit reticulocyte lysate. The translation products were analyzed on SDS-polyacrylamide gels either directly or after immunoprecipitation with human sera. Most proteins could be mapped to short defined regions of the EBV genome using short restriction fragments and overlapping sheared fragments and there is evidence of splicing for some mRNA species. The synthesis of five early proteins can be seen only with hybrid-selected RNA from induced Raji cells. These mRNAs seem to be enriched in the cells restricted to early antigen synthesis.     

Genetic variation during persistent reovirus infection: isolation of cold-sensitive and temperature-sensitive mutants from persistently infected L cells

We have examined the evolution of reovirus in two independently established persistently infected (p.i.) cell lines. We found that reovirus undergoes extensive mutation during persistent infection in L cells. However, there was no consistent pattern of virus evolution; in one p.i. cell line temperature-sensitive (ts) mutants were selected, whereas cold-sensitive (cs) mutants were isolated from the second p.i. culture. Neither the cs nor the ts mutants isolated from the carrier cultures expressed their defect at 37 degrees, the temperature at which the p.i. cells were maintained, indicating that the cs and ts phenotypes were nonselected markers. These results emphasize the point that emergence of the ts or cs mutants during persistent infection only signifies that the virus has changed; it does not necessarily imply that the particular mutant is essential for the maintenance of the persistent infection. Given the high mutation rate of viruses, and the wide spectrum of viral mutants present in carrier cultures, it is essential to distinguish the relevant changes from those which may simply represent an epiphenomenon. In the accompanying paper (R. S. Kauffman, R. Ahmed, and B. N. Fields Virology, 130, 79-87, 1983), we show that by using a genetic approach, it is possible to identify the viral gene(s) which are critical for the maintenance of persistent reovirus infection.     

Presence in infected cells of nonvirion proteins encoded by adenovirus messenger RNAs of the major late transcription regions L0 and L1

The adenovirus major late promoter functions at early and intermediate times to produce a limited set of mRNAs that appear in the cytoplasm of productively infected HeLa cells. These mRNAs may be translated in cell-free systems to produce two unrelated polypeptides of approximately 13,500 Mr (L0-13.5K and L0-13.6K) and a pair of related polypeptides of approximately 55,000 Mr (the L1-52K/55K proteins). Radiochemical protein sequence analysis of in vitro synthesized proteins has identified the N-terminal sequences of the L0-13.5K and L0-13.6K proteins (J. B. Lewis and C. W. Anderson (1983), Virology 127, 112-123). Additional sequence analyses confirmed the identification of the open reading frame for the L0-13.5K protein, and identified the ATG encoded by nucleotides 11,040 to 11,042 from the left end of the adenovirus genome as the initial codon of the L1-52K/55K protein. Antisera raised against synthetic peptides homologous to these three amino termini were used to demonstrate the presence of the L0-13.5K protein, the L0-13.6K protein, and the L1-52K/55K proteins in extracts of HeLa cells infected by adenovirus 2. The L0-13.5K protein was detected at early, intermediate, and late times after infection. The L0-13.6K and L1-52K/55K proteins were detected only at late times. Immunofluorescence microscopy indicated that the L0-13.6K protein is distributed around the periphery of the nucleus and along fibers running the length of the cell. Nonpermeabilized infected cells were stained by anti-L0-13.6K peptide serum at a single spot on the cell surface. Neither the L0-13.6K nor the L1-52K/55K proteins were detected in purified virus.     

Cell transformation by the left terminal regions of the adenovirus 40 and 41 genomes

To study the transforming capacity of the two fastidious adenoviruses 40 and 41 (Ad40 and Ad41), we have cloned the left terminal regions of their genomes. Transfection with plasmids containing these regions leads to transformation of primary baby rat kidney (BRK) cells. Cell lines derived from transformed cells show an intermediate transformed phenotype. The left terminal region was present in at least 50 copies in cells transformed by Ad41 and in 2-3 copies in Ad40-transformed cells. The integration patterns of the plasmids containing the left terminal region of Ad41 were similar in three different cell lines.