Nucleotide sequence of a cDNA clone carrying the glycoprotein gene of infectious hematopoietic necrosis virus, a fish rhabdovirus.

The nucleotide sequence of the mRNA encoding the glycoprotein of infectious hematopoietic necrosis virus was determined from a cDNA clone containing the entire coding region. The G-protein cDNA is 1,609 nucleotides long (excluding the polyadenylic acid) and encodes a protein of 508 amino acids. The predicted amino acid sequence was compared with that of the glycoprotein of the Indiana and New Jersey serotypes of vesicular stomatitis virus and with the glycoprotein of rabies virus, using a computer program which determined optimal alignment. An amino acid identity of approximately 20% was found between infectious hematopoietic necrosis virus and the two vesicular stomatitis virus serotypes and between infectious hematopoietic necrosis virus and rabies virus. The positions and sizes of the signal sequence and transmembrane domain and the possible glycosylation sites were determined.     

In vitro RNA transcription by the New Jersey serotype of vesicular stomatitis virus. I. Characterization of the mRNA species.

The in vitro RNA synthesis by the virion-associated RNA polymerase of vesicular stomatitis virus (VSV), New Jersey serotype, was compared with that of the serologically distinct Indiana serotype of VSV. The New Jersey serotype of VSV synthesized five distinct mRNA species in vitro, three of which were smaller than the corresponding species synthesized by the Indiana serotype of VSV. These included the mRNA's coding for the G, M, and NS proteins. By hybridization experiments, virtually no sequence homology was detected between the mRNA's of the two serotypes. Despite this lack of overall homology, the 12 to 18S mRNA species of both serotype contained a common 5'-terminal hexanucleotide sequence, G(5')ppp(5')A-A-C-A-G. The signicance of this finding in light of specific interactions between the two serotypes of VSV in vivo is discussed.     

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.     

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.     

Vesicular stomatitis virus NS proteins: structural similarity without extensive sequence homology.

The complete nucleotide sequence of the NS mRNA of vesicular stomatitis virus (New Jersey serotype) was established from two cDNA clones spanning the entire coding region of the mRNA. The gene is 856 nucleotides long and can code for a polypeptide of 274 amino acids. Comparison with the nucleotide sequence of the NS gene of the Indiana serotype revealed only 41% sequence homology. The deduced amino acid sequences of the NS proteins were only 32% homologous, with no identical stretches of more than five amino acids. However, at the C-terminal domain there was a conserved region of 21 amino acids with greater than 90% homology. Surprisingly, relative hydropathicity plots also demonstrated the presence of a large number of hydrophilic amino acids sequestered similarly over the N-terminal half of the protein. In addition, the total number of serine and threonine residues, presumptive phosphorylation sites, was similar and included seven serine and three threonine residues located at identical positions. It appears that during divergent evolution of these two vesicular stomatitis virus serotypes from a common ancestor, considerable mutation occurred in the main body of the gene but the overall structure of the protein was retained. The function of the NS protein in relation to the evolution of the two viruses is discussed.     

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.     

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 sequences of the mRNA''s encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions.

The complete nucleotide sequences of the vesicular stomatitis virus mRNA's encoding the glycoprotein (G) and the matrix protein (M) have been determined from cDNA clones that contain the complete coding sequences from each mRNA. The G protein mRNA is 1,665 nucleotides long, excluding polyadenylic acid, and encodes a protein of 511 amino acids including a signal peptide of 16 amino acids. G protein contains two large hydrophobic domains, one in the signal peptide and the other in the transmembrane segment near the COOH terminus. Two sites of glycosylation are predicted at amino acid residues 178 and 335. The close correspondence of the positions of these sites with the reported timing of the addition of the two oligosaccharides during synthesis of G suggests that glycosylation occurs as soon as the appropriate asparagine residues traverse the membrane of the rough endoplasmic reticulum. The mRNA encoding the vesicular stomatitis virus M protein is 831 nucleotides long, excluding polyadenylic acid, and encodes a protein of 229 amino acids. The predicted M protein sequence does not contain any long hydrophobic or nonpolar domains that might promote membrane association. The protein is rich in basic amino acids and contains a highly basic amino terminal domain. Details of construction of the nearly full-length cDNA clones are presented.     

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.     

Nucleotide sequences of the mRNA''s encoding the vesicular stomatitis virus N and NS proteins.

The complete nucleotide sequences of the vesicular stomatitis virus (VSV) mRNA's encoding the N and NS proteins have been determined from the sequences of cDNA clones. The mRNA encoding the N protein is 1,326 nucleotides long, excluding polyadenylic acid. It contains an open reading frame for translation which extends from the 5'-proximal AUG codon to encode a protein of 422 amino acids. The N and mRNA is known to contain a major ribosome binding site at the 5'-proximal AUG codon and two other minor ribosome binding sites. These secondary sites have been located unambiguously at the second and third AUG codons in the N mRNA sequence. Translational initiation at these sites, if it in fact occurs, would result in synthesis of two small proteins in a second reading frame. The VSV and mrna encoding the NS protein is 815 nucleotides long, excluding polyadenylic acid, and encodes a protein of 222 amino acids. The predicted molecular weight of the NS protein (25,110) is approximately one-half of that predicted from the mobility of NS protein on sodium dodecyl sulfate-polyacrylamide gels. Deficiency of sodium dodecyl sulfate binding to a large negatively charged domain in the NS protein could explain this anomalous electrophoretic mobility.     

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).     

Pseudotypes of vesicular stomatitis virus with the mixed coat of reticuloendotheliosis virus and vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) forms pseudotypes with envelope components of reticuloendotheliosis virus (REV). The VSV pseudotype possesses the limited host range and antigenic properties of REV. Approximately 70% of the VSV, Indiana serotype, and 45% of VSV, New Jersey serotype, produced from the REV strain T-transformed chicken bone marrow cells contain mixed envelope components of both VSV and REV. VSV pseudotypes with mixed envelope antigens can be neutralized with excess amounts of either anti-VSV antiserum or anti-REV antiserum.     

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.     

Antibodies to vesicular stomatitis virus in populations of feral swine in the United States

From 1979 to 1985, 941 feral swine (Sus scrofa) from 53 locations in 15 states were serologically tested for antibodies to vesicular stomatitis virus (VSV). Antibodies to New Jersey serotype VSV were present in 75 swine from five locations in Arkansas, Florida, Georgia, and Louisiana. Within these populations, antibody prevalences ranged from 10 to 100%. No antibodies to Indiana serotype were detected.     

Utilization of homotypic and heterotypic proteins of vesicular stomatitis virus by defective interfering particle genomes for RNA replication and virion assembly: implications for the mechanism of homologous viral interference

Defective interfering (DI) particles of Indiana serotype of vesicular stomatitis virus (VSV(Ind)) are capable of interfering with the replication of both homotypic VSV(Ind) and heterotypic New Jersey serotype (VSV(NJ)) standard virus. In contrast, DI particles from VSV(NJ) do not interfere with the replication of VSV(Ind) standard virus but do interfere with VSV(NJ) replication. The differences in the interfering activities of VSV(Ind) DI particles and VSV(NJ) DI particles against heterotypic standard virus were investigated. We examined the utilization of homotypic and heterotypic VSV proteins by DI particle genomic RNAs for replication and maturation into infectious DI particles. Here we show that the RNA-nucleocapsid protein (N) complex of one serotype does not utilize the polymerase complex (P and L) of the other serotype for RNA synthesis, while DI particle genomic RNAs of both serotypes can utilize the N, P, and L proteins of either serotype without serotypic restriction but with differing efficiencies as long as all three proteins are derived from the same serotype. The genomic RNAs of VSV(Ind) DI particles assembled and matured into DI particles by using either homotypic or heterotypic viral proteins. In contrast, VSV(NJ) DI particles could assemble only with homotypic VSV(NJ) viral proteins, although the genomic RNAs of VSV(NJ) DI particles could be replicated by using heterotypic VSV(Ind) N, P, and L proteins. Thus, we concluded that both efficient RNA replication and assembly of DI particles are required for the heterotypic interference by VSV DI particles.     

Glycoprotein exchange vectors based on vesicular stomatitis virus allow effective boosting and generation of neutralizing antibodies to a primary isolate of human immunodeficiency virus type 1

Live recombinant vesicular stomatitis viruses (VSVs) expressing foreign antigens are highly effective vaccine vectors. However, these vectors induce high-titer neutralizing antibody directed at the single VSV glycoprotein (G), and this antibody alone can prevent reinfection and boosting with the same vector. To determine if efficient boosting could be achieved by changing the G protein of the vector, we have developed two new recombinant VSV vectors based on the VSV Indiana serotype but with the G protein gene replaced with G genes from two other VSV serotypes, New Jersey and Chandipura. These G protein exchange vectors grew to titers equivalent to wild-type VSV and induced similar neutralizing titers to themselves but no cross-neutralizing antibodies to the other two serotypes. The effectiveness of these recombinant VSV vectors was illustrated in experiments in which sequential boosting of mice with the three vectors, all encoding the same primary human immunodeficiency virus (HIV) envelope protein, gave a fourfold increase in antibody titer to an oligomeric HIV envelope compared with the response in animals receiving the same vector three times. In addition, only the animals boosted with the exchange vectors produced antibodies neutralizing the autologous HIV primary isolate. These VSV envelope exchange vectors have potential as vaccines in immunizations when boosting of immune responses may be essential.     

Biological differences between vesicular stomatitis virus Indiana and New Jersey serotype glycoproteins: identification of amino acid residues modulating pH-dependent infectivity

We previously generated recombinant vesicular stomatitis viruses (VSV) based on the Indiana serotype genome which contained either the homologous glycoprotein gene from the Indiana serotype (VSIV-GI) or the heterologous glycoprotein gene from the New Jersey serotype (VSIV-GNJ). The virus expressing the GNJ gene was more pathogenic than the parental VSIV-GI virus in swine, a natural host (26). For the present study, we investigated the biological differences between the GI and GNJ proteins that may be related to the differences in pathogenesis between VSIV-GI and VSIV-GNJ. We show that the capacities of viruses with either the GNJ or GI glycoprotein to infect cultured cells differ depending on the pH. VSIV-GNJ could infect cells at acidic pHs, while the infectivity of VSIV-GI was severely reduced. VSIV-GNJ infection was also more sensitive to inhibition by ammonium chloride, indicating that the GNJ protein had a lower pH threshold for membrane fusion. We applied selective pressure to VSIV-GI by growing it at successively lower pH values and isolated variant viruses in which we identified amino acid changes that conferred low-pH-resistant infectivity. Repeated passage in cell culture at pH 6.8 resulted in the selection of a VSIV-GI variant (VSIV-6.8) that was similar to VSIV-GNJ regarding its pH- and ammonium chloride-dependent infectivity. Sequence analysis of VSIV-6.8 revealed that it had a single amino acid substitution in the amino-terminal region of the glycoprotein (F18L). This alteration was shown to be responsible for the observed phenotype by site-directed mutagenesis of a VSIV-GI full-length cDNA and analysis of the recovered engineered virus. A further adaptation of VSIV-6.8 to pHs 6.6 and 6.4 resulted in additional amino acid substitutions in areas of the glycoprotein that were not previously implicated in attachment or fusion.     

Vesicular stomatitis virus glycoprotein is a determinant of pathogenesis in swine,a natural host

There are two major serotypes of vesicular stomatitis virus (VSV), Indiana (VSIV) and New Jersey (VSNJV). We recovered recombinant VSIVs from engineered cDNAs that contained either (i) one copy of the VSIV G gene (VSIV-G(I)); (ii) two copies of the G gene, one from each serotype (VSIV-G(NJ)G(I)); or (iii) a single copy of the G(NJ) gene instead of the G(I) gene (VSIV-G(NJ)). The recombinant viruses expressed the appropriate glycoproteins, incorporated them into virions, and were neutralized by antibodies specific for VSIV (VSIV-G(I)), VSNJV (VSIV-G(NJ)), or both (VSIV-G(NJ)G(I)), according to the glycoprotein(s) they expressed. All recombinant viruses grew to similar titers in cell culture. In mice, VSIV-G(NJ) and VSIV-G(NJ)G(I) were attenuated. However, in swine, a natural host for VSV, the G(NJ) glycoprotein-containing viruses caused more severe lesions and replicated to higher titers than the parental virus, VSIV-G(I). These observations implicate the glycoprotein as a determinant of VSV virulence in a natural host and emphasize the differences in VSV pathogenesis between mice and swine.