In vitro RNA transcription by the New Jersey serotype of vesicular stomatitis virus. II. Characterization of the leader RNA.

The New Jersey serotype of vesicular stomatitis virus (VSV) was able to synthesize a small RNA (leader RNA) approximately 70 bases in length similar to the leader RNA synthesized in vitro by the genetically distinct Indiana serotype of VSV. Also, the New Jersey leader RNA contained the same 5'-terminal sequence, ppA-C-G, as the Indiana leader RNA and had a very similar base composition, with 42% AMP, 16% CMP, 18.6% GMP, and 23.4% UMP. The 3'-terminal sequence of the VSV New Jersey genome RNA was detemined and found to contain the sequence- Py-G-UOH, again the same as that of the Indiana serotype of VSV. Evidence that the New Jersey leader RNA is transcribed from the 3' end of the genome RNA was obtained from the fact that it can protect the 3'-terminal base of [3H]borohydride-labeled New Jersey genome RNA from RNase digestion. Although the New Jersey and Indiana leader RNAs were similar in many respects, they were unable to form RNase-resistant hybrids when annealed to heterologous genome RNA.     

Characterization of vesicular stomatitis virus mRNA species synthesized in vitro.

The smallest size class of mRNA (12S) synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus contains two mRNA species of similar molecular weight that code for the viral M and NS proteins. The resolution of these mRNA species was achieved by converting them to duplexes by annealing with the genome RNA, followed by RNase T2 treatment and separation in a polyacrylamide gel. Using this separation technique, the mRNA's were identified by comparing the relative resistance of their syntheses to UV irradiation of the virus. The molecular weights of these two mRNA species calculated as duplex RNAs were smaller than expected. The possible reasons for this discrepancy are discussed.     

Nucleotide sequence of the leader RNA of the New Jersey serotype of vesicular stomatitis virus.

Sequence for the leader RNA Synthesized by the New Jersey serotype of vesicular stomatitis virus is presented and its complementary sequence representing the 3'-terminal sequence of the genome RNA is deduced. Comparison with the leader RNA sequence of the serologically distinct Indiana strain reveals that the 3'-terminal region of the genomes of two viruses is highly conserved.     

Classification of the New Jersey serotype of vesicular stomatitis virus into two subtypes.

We propose a reclassification of five strains of the New Jersey serotype of vesicular stomatitis virus into two subtypes designated Concan and Hazelhurst. This subclassification into two subtypes is based on reciprocal differences in antibody neutralization of virion infectivity, nucleotide base sequence homology, oligonucleotide maps of virion RNA, and interference by defective-interfering particles.     

A unique RNA species involved in initiation of vesicular stomatitis virus RNA transcription in vitro.

Purified virions of vesicular stomatitis virus (VSV) are capable of synthesizing two distinct types of virus-specific RNA in vitro. The first consists of several viral mRNAs which have been previously shown to contain the blocked 5' terminal sequence GpppApApCpApGp and 3' terminal poly(A). The second type of RNA has an unblocked 5' terminus and does not contain poly(A) stretches long enough to bind to oligo (dT)-cellulose columns. It migrates in 20% polyacrylamide gels as a single homogeneous peak with an estimated chain length of 68 nucleotides. Base analysis demonstrated that this small RNA molecule is composed of 48% AMP, 20% CMP, 11% GMP, and 21% UMP. The 5' terminal sequence of the small RNA is ppApCpGp, which appears to be complementary to the 3' terminal sequence of the VSV genome RNA (...PypGpU). These results indicate that this small RNA molecule probably represents the intitiated lead-in RNA segment which is removed during formation of VSV mRNAs by a possible processing mechanism.     

In vitro synthesis of a unique RNA species by a T particle of vesicular stomatitis virus.

A T particle of vesicular stomatitis virus, containing most of the L-gene region, has been isolated. In vitro, these T particles synthesize exclusively a small adenine-rich RNA that is complementary to the T-particle genome. Partial sequence analysis of this small RNA indicates that it is an RNA of unique sequence with a length of approximately 45 nucleotides.     

Messenger RNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus.

The virion-associated RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV) synthesizes in vitro two size classes of RNA products similar to those observed in VSV-infected cells. One RNA product sediments at 31S with an approximate molecular weight of 2.1 X 106. The smaller products consist of at least three classes of RNA sedimenting at 17S, 14.5S, and 12S with molecular weights of 0.7 X 106, 0.52 X 106, and 0.37 X 106, respectively. Hybridization experiments show that both the 31S and 12-18S RNA products are complementary to the genome RNA, and that each class is transcribed from different nucleotide sequences. From the molecular weights of the RNA species and the hybridization experiments, it seems that almost the entire VSV genome RNA is transcribed in vitro.     

Disulfide-bonded discontinuous epitopes on the glycoprotein of vesicular stomatitis virus (New Jersey serotype).

Intrachain disulfide bonds between paired cysteines in the glycoprotein (G) of vesicular stomatitis virus (VSV) are required for the recognition of discontinuous epitopes by specific monoclonal antibodies (MAbs) (W. Keil and R. R. Wagner, Virology 170:392-407, 1989). Cleavage by Staphylococcus aureus V8 protease of the 517-amino-acid VSV-New Jersey G protein, limited to the glutamic acid at residue 110, resulted in a protein (designated GV8) with greatly retarded migration by polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions. By Western blot (immunoblot) analysis, protein GV8 was found to lose discontinuous epitope IV, which maps within the first 193 NH2-terminal amino acids. These data, coupled with those obtained by PAGE migration of a vector-expressed, truncated protein (G1-193) under reducing and nonreducing conditions, lead us to postulate the existence of a major loop structure within the first 193 NH2-terminal amino acids of the G protein, possibly anchored by a disulfide bond between cysteine 108 and cysteine 169, encompassing epitope IV. Site-directed mutants in which 10 of the 12 cysteines were individually converted to serines in vaccinia virus-based vectors expressing these single-site mutant G proteins were also constructed, each of which was then tested by immunoprecipitation for its capacity to recognize epitope-specific MAbs. These results showed that mutations in NH2-terminal cysteines 130, 174, and, to a lesser extent, 193 all resulted in the loss of neutralization epitope VIII. A mutation at NH2-terminal cysteine 130 also resulted in the loss of neutralization epitope VII, as did a mutation at cysteine 108 to a lesser extent. Both epitopes VII and VIII disappeared when mutations were made in COOH-distal cysteine 235, 240, or 273, the general map locations of epitopes VII and VIII. These studies also reveal that distal, as well as proximal, cysteine residues markedly influence the disulfide-bond secondary structure, which ostensibly determines the conformational structure of the VSV-New Jersey G protein required for presentation of the major discontinuous epitopes recognized by neutralizing MAbs.     

In vitro synthesis of a possible precursor RNA by purified vesicular stomatitis virus.

The RNA products synthesized $\text{in vitro}$ by the virion-associated RNA polymerase of purified vesicular stomatitis virus have previously been shown to contain two distinct 5′-terminal sequences. The mRNA species contain the blocked 5′-terminal G(5′)ppp(5′)A-A-C-A-G sequence and the initiated lead-in RNA segment (approximately 50 bases) contains the unblocked 5′ ppA-C-G sequence. In the present studies, using inosine 5′-triphosphate in place of GTP it is shown that RNA species as large as 14.5S contain an unblocked 5′-ppA-C-(I) sequence indicating that the GTP analogue permits synthesis of a possible precursor of viral mRNA $\text{in vitro}$.     

Molecular epizootiology and evolution of vesicular stomatitis virus New Jersey.

Vesicular stomatitis virus (VSV) has been shown previously to be capable of undergoing rapid mutational change during sequential experimental infections in various tissue culture cell systems (J. Holland, K. Spindler, F. Horodyski, E. Grabau, S. Nichol, and S. Vandepol, Science 215:1577-1585, 1982). The present study was undertaken to determine the degree of genetic diversity and evolution of the virus under natural infection conditions and to gain insight into the epizootiology of the disease. Between 1982 and 1985, numerous outbreaks of VSV of the New Jersey serotype were reported throughout regions of the United States and Mexico. A T1 RNase fingerprint analysis was performed on the RNA genomes of 43 virus isolates from areas of epizootic and enzootic virus activity. This indicates that virus populations were genetically relatively homogeneous within successive U.S. virus epizootics. The data included virus isolates from different epizootic stages, geographical locations, host animals, and host lesion sites. In contrast, only distant genome RNA T1 fingerprint similarities were observed among viruses of the different U.S. epizootics. However, Mexican viruses isolated before or concurrent with U.S. epizootics had very similar RNA genome fingerprints, suggesting that Mexico may have been the possible origin of virus initiating recent U.S. VSV New Jersey outbreaks. Comparison of T1 fingerprints of viruses with enzootic disease areas revealed a greater extent of virus genetic diversity in these areas relative to that observed in epizootic areas. The evolutionary significance of these findings and their relationship to experimental data on VSV evolution are discussed.     

Characterization of the electrophoretic mobility mutation in the N protein of the tsD1 mutant of vesicular stomatitis virus New Jersey serotype

Some isolates of the temperature sensitive mutant tsD1 of complementation group D of vesicular stomatitis virus of New Jersey serotype have a nucleocapsid (N) protein which shows an increased electrophoretic mobility on sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) when compared with wild type. Utilizing techniques involving specific chemical cleavage at tryptophan or methionine residues, as well as enzymatic cleavage with carboxypeptidases A and B, we have determined that residues near the carboxyterminus are responsible for the electrophoretic difference of the mutant protein. We have further shown that there are no differences in the tryptic peptides of the mutant compared with the wild type or a non-ts revertant in this region of the protein. We have identified a tryptic peptide located outside the relevant carboxyterminal region which is distinct in mutant and revertant. We conclude that the mutation producing the aberrant electrophoretic mobility of N protein of the tsD1 mutant is a missense point mutation located at least 40 amino acid residues from the carboxyterminus and which interacts with a more proximal carboxyregion so as to influence electrophoretic mobility on SDS-PAGE.     

Nucleotide sequence of a cDNA clone encoding the entire glycoprotein from the New Jersey serotype of vesicular stomatitis virus.

The nucleotide sequence of the mRNA encoding the glycoprotein from the New Jersey serotype of vesicular stomatitis virus (VSV) was determined from a cDNA clone containing the entire coding region. The sequence of 12 5'-terminal noncoding nucleotides present in the mRNA but not in the cDNA clone was determined from a primer extended to the 5' terminus of the mRNA. The mRNA is 1,573 nucleotides long (excluding polyadenylic acid) and encodes a protein of 517 amino acids. Only six nucleotides occur between the translation termination codon and the polyadenylic acid. Short homologies between the untranslated termini of this mRNA and the mRNAs of the Indiana serotype were found. The predicted protein sequence was compared with that of the glycoprotein of the Indiana serotype of VSV and with the glycoprotein of rabies virus, using a computer program which determines optimal alignment. An amino acid identity of 50.9% was found for the two VSV serotypes. Approximately 20% identity was found between the rabies virus and VSV New Jersey glycoproteins. The positions and sizes of the transmembrane domains, the signal sequences, and the glycosylation sites are identical in both VSV serotypes. Two of five serine residues which were possible esterification sites for palmitate in the glycoprotein from the Indiana serotype are changed to glycine residues in the glycoprotein from the New Jersey serotype. Because the glycoprotein of the New Jersey serotype does not contain esterified palmitate, we suggest that one or both of these residues are the probable esterification sites in the glycoprotein from the Indiana serotype.     

Characterization and translation of methylated and unmethylated vesicular stomatitis virus mRNA synthesized in vitro by ribonucleoprotein particles from vesicular stomatitis virus-infected L cells.

Ribonucleoprotein particles isolated from extracts of vesicular stomatitis virus (VSV) -infected L cells synthesized in vitro four classes of polyadenylated RNA sedimenting at 29S, 19S, 17S, and 13S. When synthesized in vitro in the presence of the methyl donor S-adenosyl methionine, these RNA species contained the following 5'-terminal structures: (i) m7G5ppp5'AmpAp(70%) ; (ii) m7G5'ppp5'AmpAmpNp (20%) and (iii) pppAp (10%). In the presence of the methylation inhibitor S-adenosylhomocysteine, however, the mRNA contained the 5'-terminal structures G5'ppp5'Ap (80%) and pppAp (20%). The mRNA's synthesized in vitro were translated in the homologous ascites and the heterologous wheat embryo cell-free systems. In both, the products were shown by sodium dodecyl sulfate gel electrophoresis and by immunoprecipitation to contain all five viral proteins, L, G, N, NS, and M. The presumed precursor to the G protein (G*) was also identified by fingerprint analysis. Methylated VSV mRNA was more active in protein synthesis than unmethylated mRNA in both the ascites system and the wheat embryo systems. Addition of S-adenosylmethionine stimulated translation of unmethylated mRNA in the wheat embryo but not in the ascites extract. S-adenosylhomocysteine, however, by preventing mRNA methylation inhibited the translation of unmethylated VSV mRNA in both systems. The mRNA methylating activity present in wheat embryo S-30 extracts was recovered in the ribosome-free supernatant fraction (S-150) and was insensitive to the protein synthesis inhibitor pactamycin.     

Genetic diversity of enzootic isolates of vesicular stomatitis virus New Jersey.

The RNA genomes of 43 vesicular stomatitis virus (VSV) isolates of the New Jersey (NJ) serotype were T1-ribonuclease fingerprinted to compare the extent of genetic diversity of virus from regions of epizootic and enzootic disease activity. Forty of these viruses were obtained from Central America during 1982 to 1985. The other three were older isolates, including a 1970 isolate from Culex nigripalpus mosquitos in Guatemala, a 1960 bovine isolate from Panama, and a 1976 isolate from mosquitos (Mansonia indubitans) in Ecuador. The data indicate that extensive genetic diversity exists among virus isolates from this predominantly enzootic disease zone. Six distinct T1 fingerprint groups were identified for the Central American VSV NJ isolates from 1982 to 1985. The 1960 VSV NJ isolate from Panama and the 1976 isolate from Ecuador formed two additional distinct fingerprint groups. This finding is in sharp contrast to the relatively close genetic relationship existing among VSV NJ isolates obtained from predominantly epizootic disease areas of the United States and Mexico during the same period (S. T. Nichol, J. Virol. 61:1029-1036, 1987). In this previous study, RNA genome T1 fingerprint differences were observed among isolates from different epizootics; however, the isolates were all clearly members of one large T1 fingerprint group. The eight T1 fingerprint groups described here for Central American and Ecuadorian viruses are distinct from those characterized earlier for virus isolates from the United States and Mexico and for the common laboratory virus strains Ogden and Hazelhurst. Despite being isolated 14 years earlier, the 1970 insect isolate from Guatemala is clearly a member of one of the 1982 to 1985 Central American virus fingerprint groups. This indicates that although virus genetic diversity in the region is extensive, under certain natural conditions particular virus genotypes can be relatively stably maintained for an extended period. The implications of these findings for the evolution of VSV NJ and epizootiology of the disease are discussed.