Robert R. Wagner

Reproduction of Rhabdoviruses

The rhabdoviruses are ubiquitous, highly infectious agents of animal and plant disease and are generally transmitted by arthropods. Assignment of viruses to the taxon rhabdoviruses (rod-shaped viruses) was originally based entirely on morphology. This classification has turned out to be fortuitously fortunate because later biochemical studies have revealed remarkable uniformity among these structurally similar viruses isolated from extremely diverse hosts. It is perhaps not farfetched to postulate a common ancestor for all the rhabdoviruses of plants, arthropods, and vertebrates. Classification of a virus as a rhabdovirus should be based on the following most important characteristics: n n1. n nRhabdoviruses are rod-shaped particles, varying considerably in length (60–400 nm) but of a reasonably consistent width (60–85 nm). n n n n n2. n nAnimal rhabdoviruses tend to be bullet-shaped in appearance, flat at one end and a tapered sphere at the other. Plant rhabdoviruses are usually bacilliform in shape, quite elongated and with two round ends. n n n n n3. n nAll rhabdoviruses appear to be surrounded by a membranous envelope with protruding spikes. All these viruses probably contain lipids and are, therefore, susceptible to disruption by ether and detergents.

The plus-strand leader RNA of VSV inhibits DNA-dependent transcription of adenovirus and SV40 genes in a soluble whole-cell extract

In an attempt to determine the mechanism (or mechanisms) by which vesicular stomatitis virus (VSV) kills cells, products of VSV transcription were tested in a cell-free system for their capacity to inhibit transcription of SV40 DNA and plasmids containing adenovirus late promoter and adenovirus-associated RNA genes. VSV RNA transcripts and other RNAs were compared for their capacity to suppress transcription of these DNA templates by RNA polymerases and cofactors present in the HeLa-cell extract system. Relatively low concentrations of the plus-strand leader RNA made in vitro from the 3 end of the wild-type VSV genome were found to inhibit initiation of transcription catalyzed by both RNA polymerase II and RNA polymerase III. Polyadenylated VSV messengers and other natural and synthetic RNAs also caused some inhibitory effects on in vitro transcription from DNA templates, but only at extremely high concentrations. Compared with the wild-type plus-strand RNA leader, the leader RNA synthesized in vitro by defective-interfering VSV showed only limited capacity to inhibit RNA synthesis on adenovirus and SV40 DNA templates and only at concentrations at least 30 times greater than that of the wild-type leader. The existence of nucleotide sequences in wild-type leader RNA, not present in defective-interfering leader RNA, that could recognize and block promoters, polymerases or protein cofactors is discussed.

Point mutations in glycoprotein gene of vesicular stomatitis virus (New Jersey serotype) selected by resistance to neutralization by epitope-specific monoclonal antibodies

Antigenic variants of the New Jersey serotype of vesicular stomatitis virus (VSV-NJ) were isolated and cloned by selecting virus plaques resistant to neutralization by high-titered monoclonal antibodies (MAbs) directed to glycoprotein (G) epitopes V, VI, VII, or VIII. The G proteins of each neutralization-resistant virus variant also exhibited markedly reduced antigenic reactivity with each corresponding epitope-specific MAb as determined by enzyme-linked immuno-absorbent assay and by Western blot analysis. Loss of antigenic reactivity of certain mutant G proteins to a MAb other than the one used to select the mutant virus suggested close antigenic proximity, particularly for epitopes VI and VII. The virion RNAs coding for the entire G gene of the wild-type virus and 10 MAb-induced mutants were sequenced by primer DNA extension using the dideoxy method. Each mutant G gene exhibited only a single nucleotide change, leading in each case to a single amino acid substitution, as follows: Glu210----Lys for all three mutants selected by MAb14 (epitope VII); Pro268----Thr for one mutant selected by MAb12 (epitope VI); Ser277----Lys for all three mutants selected by MAb15 (epitope VIII); and Glu364----Lys for all three mutants selected by MAb11 (epitope V). These neutralizing MAb-selected mutations are clustered in the middle third of the 517-amino acid VSV-NJ G protein, presumably resulting in conformational changes that alter recognition of one or more antigenic determinants by a specific monoclonal antibody.

Glycoprotein micelles isolated from vesicular stomatitis virus spontaneously partition into sonicated phosphatidylcholine vesicles

Abstract Micelles formed by glycoprotein of vesicular stomatitis virus, isolated by octylglucoside extraction and purified free of detergent and phospholipid, spontaneously partitioned into preformed sonicated egg phosphatidylcholine vesicles when the glycoprotein rosettes and vesicles were incubated together in 10 m M Tris (pH 7.5) at 37°. The hydrophobic tail fragment of the glycoprotein became resistant to protease digestion when the glycoprotein was inserted into the vesicle bilayer. When the covalently attached fatty acid of the glycoprotein was labeled in vivo with [3H]lpalmitate, it was demonstrated that the fatty acid was attached to the hydrophobic glycoprotein tail fragment in vesicle and virion membranes.

Epitope mapping by deletion mutants and chimeras of two vesicular stomatitis virus glycoprotein genes expressed by a vaccinia virus vector

Deletion mutants and chimeras of the glycoprotein (G) genes of vesicular stomatitis virus serotypes Indiana (VSV-Ind) and New Jersey (VSV-NJ) were cloned in plasmids and vaccinia virus vectors under control of the bacteriophage T7 polymerase promoter for expression in CV-1 cells co-infected with a T7 polymerase-expressing vaccinia virus recombinant. Truncated and chimeric G proteins expressed by these vectors were tested for their capacity to react with VSV-Ind and VSV-NJ epitope-specific monoclonal antibodies (MAbs) by Western blot analysis for those antigenic determinants not affected by disulfide-bond reducing conditions or by immuno dotblot analysis for those that are. These experiments allowed us to create putative epitope maps for glycoproteins of both serotypes based on binding affinity and cross-reactivity of VSV-Ind and VSV-NJ MAbs for truncated or chimeric G proteins of known amino acid sequences. Seven of the 9 VSV-NJ G epitopes, including all 4 epitopes involved in virus neutralization by MAbs, mapped to the center (amino acid sequence 193-289) of the 517 amino acid VSV-NJ G protein. Four of the 11 VSV-Ind G epitopes, including 2 neutralizable epitopes, mapped to the cysteine-rich amino-terminal domain (amino acid sequence 80-183) of the 511 amino acid VSV-Ind G protein; the remaining 7 VSV-Ind G epitopes, including 2 involved in virus neutralization, were clustered in the cysteine-poor carboxy-terminal domain (amino acid sequence 286-428). In site-specific mutants of the VSV-Ind G gene defective in one or both glycosylation sites, only the amino-terminal epitopes of the VSV-Ind G protein were affected by deletion of the carbohydrate chain at residue 179; deletion of the carbohydrate chain at residue 336 did not alter reactivity of the G protein with any of the relevant monoclonal antibodies. These results are discussed in relation to earlier attempts to map the antigenic determinants of VSV-NJ and VSV-Ind G proteins by proteolysis of the G protein and by sequencing the G genes of mutant viruses selected for their resistance to neutralization by epitope-specific monoclonal antibodies.

Monoclonal antibodies to the glycoprotein of vesicular stomatitis virus (New Jersey serotype): A method for preliminary mapping of epitopes

Of the nine antigenic determinants on the glycoprotein (G) of the New Jersey serotype of vesicular stomatitis virus (VSV) identified by competitive binding of 25 monoclonal antibodies (MAbs), those relegated to epitopes I, II, III, and IV exhibited no significant ability to neutralize virus infectivity but some nonneutralizing MAbs cross-reacted by ELISA with the G protein of VSV-Indiana. High-titered neutralization of homotypic virus was exhibited by epitope V, VI, and VII MAbs but quite variable neutralizing activity was found among MAbs of epitope family VIII and particularly the heterogeneous epitope family IX. Peptide mapping of the epitopes was not feasible because most MAbs would not bind by Western blotting to G protein under standard conditions of proteolysis or disulfide bond reduction. Therefore, a technique was devised for roughly locating epitopes by protease footprinting of G protein partially protected by individual MAbs complexed with staphylococcal protein A-Sepharose beads. Under these conditions, MAbs to all nine epitopes protected a similar 12-kDa fragment of the G protein from proteolysis by Staphylococcus aureus V8 protease. N-Terminal amino acid sequencing mapped two of these 12-kDa peptide footprints to a position on the G protein extending from amino acid 219 to about 100 amino acids downstream. Although MAbs to only one epitope bound to the 12-kDa fragment by Western blotting, these data suggest, but do not prove, that all nine epitopes of the undenatured VSV-New Jersey G protein are clustered at the middle 20% of a highly structured protein. This method may help to identify the general regions for epitopes on complex proteins of as yet unknown three-dimensional structure.

Envelope proteins of vesicular stomatitis virions: Accessibility to iodination

Abstract The glycoprotein and, to a lesser extent, the matrix membrane protein of intact vesicular stomatitis virions were specificially iodinated by oxidation with lactoperoxidase or chloramine T. The virion envelope provided an effective barrier against iodination of nucleocapsid proteins. Selective removal of glycoprotein by trypsin or Triton X-100 exposed the membrane matrix protein to somewhat more extensive iodination but the nucleocapsid N protein became only slightly more accessible to iodination; the nucleocapsid L and NS proteins remained unlabeled.

Nucleotide sequence and secondary structure of VSV leader RNA and homologous DNA involved in inhibition of DNA-dependent transcription

We have analyzed the nucleotide sequences and secondary structure required for the transcriptional inhibitory activity of the plus-strand leader RNA of vesicular stomatitis virus (VSV) in a reconstituted HeLa cell transcription system using the adenovirus-2 late promoter (LP) and virus-associated (VA) genes as templates. The New Jersey serotype (VSVNJ) leader and the leader of the Indiana serotype (VSVInd) both contain cleavage sites for the double-strand-specific ribonuclease V1, and these sites are consistent with the presence of a predicted AU-rich stem-loop structure. Studies in which the secondary structure was perturbed with the intercalating agent proflavin suggested that a stem-loop structure enhances the efficiency of transcription inhibition in the VSVNJ leader. Experiments using leader RNA fragments, a VSVInd cDNA derived from the 3 end of the genome, and synthetic oligodeoxynucleotide homologous to regions of the VSV leader indicated that the AU(AT)-rich center region of the VSV leader molecule is sufficient to inhibit DNA-dependent transcription directed by both polymerase II and III, but flanking nucleotide sequences are important for more efficient inhibition of transcription.

The structure of vesicular stomatitis virus membrane A phosphorus nuclear magnetic resonance approach

The proton decoupled 40.48 M Hz 31P NMR spectrum of intact and unperturbed membrane-enclosed vesicular stomatitis virus (sterotype Indiana) exhibited two distinct maxima. These can be resolved into a narrow, symmetric line and a broad asymmetric line. The 31P NMR spectrum of a multilamellar (unsonicated) preparation of the extracted viral lipids exhibited a line shape similar to that of the intact virus. A sonicated vesicle preparation of the extracted viral lipids exhibited a narrow symmetric line. The narrow component in the intact virus spectrum may be attributed to small membrane fragments. Phospholipase C digestion of the intact virus resulted in substantial reduction in intensity of both components which suggests that much of the contribution to both peaks is due to phosphate in the phospholipid polar head groups. The phospholipid phosphates in both sonicated and unsonicated preparations of the extracted viral lipids exhibited substantially longer relaxation times than did those in the intact virus. The short relaxation time emanating from the intact virus preparation is caused by immobilization of the phospholipid head groups which could be due to lipid-protein interactions. Trypsin treatment of vesicular stomatitis virions, which results in complete removal of the exterior hydrophilic segment of the membrane glycoprotein, increased the 31P relaxation time to a value similar to that observed in the protein-free total lipid extracts; this finding provides supporting evidence for the role of virus glycoprotein in shortened relaxation times. A reversible temperature-dependent change in apparent line width and absence of an effect of cholesterol on the 31P phospholipid spectrum were also demonstrated.

Lipid and protein contributions to the membrane surface potential of vesicular stomatitis virus probed by a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin

The surface potential of membranes of vesicular stomatitis virus and liposomes was determined by shift of ionization over a wide pH range of the membrane-inserted fluorophore, 4-heptadecyl-7-hydroxycoumarin. Incorporation into sonicated vesicles of negatively charged phosphatidylserine markedly increased the surface potential of uncharged phosphatidylcholine, but no significant effect on surface potential was produced by polar but uncharged glucocerebroside incorporated in phosphatidylcholine vesicles. The membrane of vesicular stomatitis virus was found to have a moderately high surface potential. Contributing to this viral membrane surface potential were glycoprotein spikes and phospholipid headgroups as determined by lowered charge after treatment of intact virions with thermolysin to remove glycoprotein or phospholipase C to remove phospholipid headgroups. The role of viral glycoprotein was confirmed by demonstrating increased surface charge of vesicles reconstituted with both viral glycoprotein and lipids compared with vesicles reconstituted with viral lipids alone. An unexpected finding was the large contribution to surface potential of cholesterol present in viral membrane. Increasing cholesterol concentration in virions by interaction with cholesterol-complexed serum lipoproteins resulted in a marked decrease in surface potential, whereas 75% depletion of virion cholesterol by interaction with sphingomyelin-complexed serum lipoproteins resulted in a significant increase in virion membrane surface potential. Although removal of glycoprotein spikes or depletion of cholesterol causes reduction in infectivity of vesicular stomatitis virus, no direct correlation could be found between alteration in surface charge and infectivity.