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.     

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.     

Alfalfa mosaic virus temperature-sensitive mutants. I. Mutants defective in viral RNA and protein synthesis

The replication in cowpea protoplasts of temperature-sensitive (ts) mutants of alfalfa mosaic virus (AIMV) was studied at the permissive (25 degrees) and the restrictive (30 degrees) temperature. Using the Northern blot hybridization technique, it was shown that at the restrictive temperature two RNA 1 mutants, Bts 03 and Bts 04, and two RNA 2 mutants, Mts 03 and Mts 04, were all defective in the synthesis of viral minus-strand RNA, whereas the synthesis of the plus-strand genomic RNAs 1, 2, and 3 and the subgenomic coat protein messenger, RNA 4, was relatively unimpaired. In Bts 04 inoculated protoplasts the RNA 4 produced at 30 degrees was translated into coat protein and viral RNA was encapsidated to give infectious virus. RNA 4 in Bts 03 and Mts 04 infected protoplasts was not translated into coat protein at 30 and consequently there was no assembly of infectious virus. Protein synthesis by Mts 03 was not investigated. A1MV RNAs 1 and 2 encoded proteins are both involved in the synthesis of viral minus-strand RNA and the translation of RNA 4 and possibly other viral messengers. The results with Bts 03 and Bts 04 show that the two functions of the RNA 1 encoded protein can be mutated separately.     

Inhibition of SV40 DNA synthesis by vesicular stomatitis virus in doubly infected monkey kidney cells

Vesicular stomatitis virus (VSV) inhibited SV40 DNA synthesis in doubly infected synchronized Vero cells. Gel-electrophoretic profiles demonstrated that SV40 DNA monomers accumulated in all stages of supercoiling, regardless of whether cells were superinfected with VSV early or late in the S phase. These gel profiles were indistinguishable from ones obtained from SV40-infected, cycloheximide-treated cells in the absence of VSV infection. Radiolabel in the partial supercoils could be chased into supercoils, but only by restoring protein synthesis. The relative rates of SV40 DNA chain elongation were determined in VSV-superinfected and nonsuperinfected cells. The gradients of 3H incorporation as a function of distance from the origin of replication in pulse-labeled form I DNA were unaffected by VSV. It is concluded that VSV inhibition of SV40 DNA synthesis is an indirect result of inhibition of host cell protein synthesis and it is suggested that incompletely supercoiled SV40 chromatin is not a good template for DNA synthesis.     

Virus protein synthesis in alfalfa mosaic virus infected alfalfa protoplasts

Four proteins unique to virus infection were synthesized in alfalfa mosaic virus-infected alfalfa mesophyll protoplasts. These proteins, P1, P2, P3, and coat protein comigrated on electrophoresis with the major in vitro translation products of RNA 1, RNA 2, RNA 3, and RNA 4, respectively. P1, P3, and coat protein were observed at 5 hr post inoculation; P2 was detected at 9 hr post inoculation. The three nonstructural proteins accumulated most rapidly early in infection until about 15 hr post inoculation; stable protein levels were maintained thereafter. Coat protein accumulated rapidly until about 20 hr after inoculation. All four virus RNA species were detected in infected protoplasts by labelling with [3H]uridine. Ultraviolet irradiation of protoplasts prior to inoculation was necessary for virus protein detection, but it severely depressed the synthesis of RNA 1 and RNA 2 relative to RNA 3 and RNA 4.     

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.     

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.     

Cellular pools of viral proteins in 3T3 cells chronically infected with Moloney-murine leukemia virus

The kinetics of incorporation of [³H]leucine into proteins of mature Moloney-MuLV was followed to estimate the cellular pools of the viral proteins, and their precursors in 3T3 cells chronically infected with M-MuLV. Viruses were isolated by isopycnic density gradient centrifugation, and their proteins were separated by SDS-polyacrylamide gel electrophoresis. Entrance of labelled proteins into complete virions reached the maximal rate at different times after addition of labelled amino acids. A 33,000 dalton protein (33k) was the first and p30 was the second to enter complete virions 1.5 to 2 hr after introduction of label. A 19,000-dalton protein and gp72 were the last, reaching maximal values for entrance into virions 6 hr after labelling. These lag periods indicate that the budding process of virions takes less than 1.5 hr, whereas modification plus assembly of some proteins into mature virions takes 2–4 hr in exponentially growing cells. The more rapid appearance of label in 33k raises the question as to whether this protein has a regulatory role in virus formation.     

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.     

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.