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.     

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.     

Terminal sequences of vesicular stomatitis virus RNA are both complementary and conserved.

The nucleotide sequences at the 5' and 3' termini of RNA isolated from the New Jersey serotype of vesicular stomatitis virus [vsV(NJ)] and two of its defective interfering (DI) particles have been determined. The sequence differs from that previously demonstrated for the RNA from the Indiana serotype of VSV at only 1 of the first 17 positions from the 3' terminus and at only 2 of the first 17 positions from the 5' terminus. The 5'-terminal sequence of VSV(NJ) RNA is the complement of the 3'-terminal sequence, and duplexes which are 20 bases long and contain the 3' and 5' termini have been isolated from this RNA. The RNAs isolated from DI particles of VSV(NJ) have the same base sequences as do the RNAs from the parental virus. These results are in sharp contrast to those obtained with the Indiana serotype of VSV and its DI particles, in which the 3'-terminal sequences differ in 3 positions within the first 17. However, with both serotypes, the 3'-terminal sequence of the DI RNA is the complement of the 5'-terminal sequence of the RNA from the infectious virus. These findings suggest that the 3' and 5' RNA termini are highly conserved in both serotypes and that the 3' terminus of DI RNA is ultimately derived by copying the 5' end of the VSV genome, as recently proposed (D. Kolakofsky, M. Leppert, and L. Kort, in B. W. J. Mahy and R. D. Barry, ed., Negative-Strand Virus and the Host Cell, 1977; M. Leppert, L. Kort, and D. Kolakofsky, Cell 12:539-552, 1977; A. S. Huang, Bacteriol. Rev. 41:811-8218 1977).     

Oligonucleotide fingerprints of RNA species obtained from rhabdoviruses belonging to the vesicular stomatitis virus subgroup.

The relationships among the genomes of various rhabdoviruses belonging to the vesicular stomatitis virus subgroup were analyzed by an oligonucleotide fingerprinting technique. Of 10 vesicular stomatitis viruses, Indiana serotype (VSV Indiana), obtained from various sources, either no, few, or many differences were observed in the oligonucleotide fingerprints of the 42S RNA species extracted from standard B virions. Analyses of the oligonucleotides obtained from RNA extracted from three separate preparations of VSV Indiana defective T particles showed that their RNAs contain fewer oligonucleotides than the corresponding B particle RNA species. The fingerprints of RNA obtained from five VSV New Jersey serotype viruses were easily distinguished from those of the VSV Indiana isolates. Three of the VSV New Jersey RNA fingerprints were similar to each other but quite different from those of the other two viruses. The RNA fingerprints of two Chandipura virus isolates (one obtained from India and one from Nigeria) were also unique, whereas the fingerprint of Cocal virus RNA was unlike that of the serologically related VSV Indiana.     

Comparisons of nucleotide sequences in the genomes of the New Jersey and Indiana serotypes of vesicular stomatitis virus.

Nucleotide sequences of around 200 residues were determined adjacent to the 3' terminus of the genome RNA of vesicular stomatitis virus, New Jersey serotype, and adjacent to the 3'-terminal polyadenylic acid tract of the N protein mRNA of the same virus. These sequences were compared with the corresponding sequences previously determined for the Indiana serotype of vesicular stomatitis virus. The sequences obtained for the two strains were readily aligned, showing 70.8% homology overall. Examination of the sequences allowed identification of the translation initiation and termination codons for the N mRNA of each serotype. The deduced N-terminal and C-terminal amino acid sequences of the two N polypeptides were each similar, and most of the differences between them consisted of substitution by a clearly homologous amino acid. It was proposed that these nucleotide sequences, within limits imposed by their functions, comprise reasonably representative measures of the extent of sequence homology between the genomes of the two serotypes, and that this is higher than previously estimated, but with little exact homology over extended regions.     

Polymerase errors accumulating during natural evolution of the glycoprotein gene of vesicular stomatitis virus Indiana serotype isolates

We report the entire glycoprotein (G) gene nucleotide sequences of 26 vesicular stomatitis virus Indiana serotype (VSV IND) type 1 isolates from North and Central America. These sequences are also compared with partial G gene sequences of VSV IND type 2 (Cocal) and type 3 (Alagoas) viruses and the complete G gene sequences of the more distantly related VSV New Jersey (NJ) and Chandipura viruses. Phylogenetic analysis of the G gene sequences by maximum parsimony revealed four major lineages or subtypes within the classical VSV IND (type 1) viruses, each with a distinct geographic distribution. A high degree of VSV genetic diversity was found in Central America, with several virus subtypes of both VSV IND and NJ serotypes existing in this mainly enzootic disease region. Nineteen percent sequence variation but no deletions or insertions were evident within the 5' noncoding and the coding regions of the VSV IND type 1 G genes. In addition to numerous base substitutions, the 3' noncoding regions of these viruses also contained numerous base insertions and deletions. This resulted in striking variation in G gene sizes, with gene lengths ranging from 1,652 to 1,868 nucleotides. As the VSV IND type 1 subtypes have diverged from the common ancestor with the NJ subtypes, their G mRNAs have accumulated more 3' noncoding sequence inserts, ranging up to 303 nucleotides in length. These primarily consist of an imprecise reiteration of the sequence UUUUUAA, apparently generated by a unique polymerase stuttering error. Analysis of the deduced amino acid sequence differences among VSV IND type 1 viruses revealed numerous substitutions within defined antigenic epitopes, suggesting that immune selection may play a role in the evolution of these viruses.     

Two methyltransferase activities in the purified virions of vesicular stomatitis virus.

In addition to an RNA-dependent RNA polymerase, purified vesicular stomatitis virus contains a methyltransferase activity which transfers the methyl group from the methyl donor, S-adenosyl-L-methionine, to two positions in the 5'-terminal capped structure of the nascent mRNA's synthesized in vitro as 7mG-(5)'ppp(5')Apm... In the present study it is shown that two distinct methyltransferase activities are discernible in the purified virus. The in vitro concentrations of the methyl donor specify the number and location of the methyl groups transferred to the capped 5'-termini of VSV mRNA's. Limited concentrations of the methyl donor result in a single methylation of the penultimate base in the 2'-hydroxyl position, that is, G(5')ppp(5')Apm..., whereas saturating concentrations of the methyl donor methylate the blocking guanosine residue at the 7-position, resulting in the dimethylated cap, 7mG(5')ppp(5')Apm... Pulse-chase experiments demonstrate that the monomethylated cap structure is the precursor substrate for the dimethylated cap. In this respect, vesicular stomatitis virus system is quite distinct from the vaccinia and reovirus systems. Virus purified from different host cells including hamster, mouse, and human contain both methyltransferase activities. The mRNA's containing monomethylated capped structures are poor templates for protein synthesis in vitro.     

RNA synthesis of vesicular stomatitis virus. VIII. Oligonucleotides of the structural genes and mRNA.

The single-stranded RNA genome of vesicular stomatitis virus (VSV, Indiana serotype, San Juan strain) yields approx. 75 RNase T1-resistant oligonucleotides ranging in size from 10 to 50 bases. Each of the five structural genes, isolated as duplex RNA molecules hybridized to complementary mRNA, contains two or more of these large oligonucleotides. One of the oligonucleotides is identified as part of the non-coding region near the 3' end of the genome. Comparison of these results with others indicate that the RNA sequence of VSV is apparently stable in the laboratory but not in the wild. RNase T1-resistant oligonucleotides are also shown for all five VSV mRN species. Whether the mRNA for these digestions are are isolated from duplex RNA molecules or as single-stranded RNA species, the oligonucleotide patterns for each mRNA are virtually identical, indicating that each mRNA is transcribed from contiguous sequences on the genome. Comparison with published oligonucleotide patterns obtained from other isolates of VSV or from VSV deletion mutants indicate that identity and changes in their genome structure can be correlated with specific structural genes.     

Proteins of Vesicular Stomatitis Virus: II. Immunological Comparisons of Viral Antigens

The antigens of the nucleoprotein core and the coat of vesicular stomatitis virus (VSV) particles of the Indiana serotype were prepared and purified by sucrose gradient fractionation. Antibody was prepared separately to each of the two antigen fractions. By immunological procedures, it was shown that soluble antigens of VSV preparations sedimenting at 20S and in the leading edge of the 6S region are antigenically related to VP3, the protein of the virus core, whereas the 6S soluble antigen cross-reacts only with viral coat antibodies. These results confirm previous results obtained by polyacrylamide gel analysis of the antigens. It has further been demonstrated that the 6S antigen is a glycoprotein. Comparing antigens of the New Jersey and Indiana serotype showed that the coat antigens of virus particles and the 6S antigen are immunologically distinct in the two serotypes. In complement-fixation tests, the core antigens and the soluble 20S antigens from one serotype showed a cross-reaction with antiserum prepared against core proteins of the other serotype.     

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.     

Restitution of infectivity to spikeless vesicular stomatitis virus by solubilized viral components.

Noninfectious spikeless particles have been obtained from vesicular stomatitis virus (VSV, Indiana serotype) by bromelain or Pronase treatment. They lack the viral glycoprotein (G) but contain all the other viral components (RNA, lipid, and other structural proteins). Triton-solubilized VSV-Indiana glycoprotein preparations, containing the viral G protein as well as lipids (including phospholipids), have been extracted from whole virus preparations, freed from the majority of the detergent, and used to restore infectivity to spikeless VSV. The infectivity of such particles has been found to be enhanced by poly-L-ornithine but inhibited by Trition or homologous antiserum pretreatment. Heat-denatured glycoprotein preparations were not effective in restoring the infectivity to spikeless VSV. Heterologous glycoprotein preparations from the serologically distinct VSV-New Jersey serotype were equally capable of making infectious entities with VSV-Indiana spikeless particles, and the infectivity of these structures was inhibited by VSV-New Jersey antiserum but not by VSV-Indiana antiserum. Purified, detergent-free glycoprotein selectively solubilized from VSV-Indiana by the dialyzable detergent, octylglucoside, also restored infectivity of spikeless virions of VSV-Indiana and VSV-New Jersey.     

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.     

Assignment of the large oligonucleotides of vesicular stomatitis virus to the N, NS, M, G, and L genes and oligonucleotide gene ordering within the L gene.

Analyses of prototype vesicular stomatitis (VSV, Indiana serotype) mRNA-32P-labeled viral RNA duplexes have established the assignments of 65 of the 72 large oligonucleotides that are recovered by two-dimensional electrophoresis of RNase T1 digests of the viral RNA. Fifty of the oligonucleotides are recovered in the L RNA duplex, four each in the N, M, and NS duplexes, and three in the G RNA duplex. Studies of three small defective-particle RNA species indicate that they have only L gene oligonucleotides in addition to three of the seven unassigned oligonucleotides. Some L gene ordering of oligonucleotides can be postulated from the defective-particle RNA sequence analyses. Analyses of naturally occurring alternate isolates of VSV Indiana have established that by comparison to the prototype virus strain, the alternate isolates minimally have genome sequence differences in L, G, N, NS and/or unassigned regions of the genome. Changes in the genome have also been induced by vitro high-level mutagenesis of the prototype virus.