Indiana Serotype Of Vesicular Stomatitis Virus Research Articles

Expression of L protein of vesicular stomatitis virus Indiana serotype from recombinant baculovirus in insect cells: requirement of a host factor(s) for its biological activity in vitro.

The 241-kDa large (L) protein of vesicular stomatitis virus (VSV) Indiana serotype, a multifunctional catalytic subunit of the viral RNA polymerase, has been expressed in Spodoptera frugiperda cells infected with recombinant baculovirus BacPAK6-L containing the L gene under the control of a polyhedrin promoter. The recombinant L protein was biologically active and supported viral mRNA synthesis in vitro. When the expressed L protein was purified by phosphocellulose column chromatography, it eluted in two peaks, one at 0.4 M NaCl (peak I) and the second at 0.75 M NaCl (peak II). The L protein in peak I showed significant transcriptional activity in an in vitro transcription reconstitution experiment, whereas the L protein in peak II was inactive. Interestingly, the addition of cytoplasmic extract from uninfected Sf21 cells to peak II completely restored transcription in vitro, indicating the requirement of a host factor(s) for the activity of the L protein. This factor is relatively heat stable and is dissociable from the recombinant L protein. It is also present in BHK, COS, and HeLa cells in detectable levels. The role of the putative host protein(s) in the activation of the L protein is discussed.

Identification of a Set of Proteins (C′ and C) Encoded by the Bicistronic P Gene of the Indiana Serotype of Vesicular Stomatitis Virus and Analysis of Their Effect on Transcription by the Viral RNA Polymerase

Previous work (C. F. Spiropoulou and S. T. Nichol, 1993,J. Virol.67, 3103–3110) has demonstrated the existence in cells infected with the New Jersey serotype of vesicular stomatitis virus (VSV) of two small carboxy-coterminal proteins encoded by the P mRNA. These proteins have been named C′ and C. We are interested in studying the function of these proteins in the virus life cycle. Toward this end, we have cloned the ORF encoding the potential C′ protein of the Indiana serotype as a histidine-tagged fusion protein, purified the expressed protein fromEscherichia coli,and used the fusion protein as an immunogen to raise antiserum in a rabbit. We have used this anti-C′ protein serum to demonstrate that both of the predicted C′ and C proteins are synthesized in cells infected with the Indiana serotype of VSV. In addition we have localized a portion of these proteins to nucleocapsids isolated from infected cells, suggesting that they may play a role in RNA synthesis. Reconstitution of the viral polymerase activity by expressing the L and P protein subunits with or without the C proteins failed to demonstrate any effect of the presence of these latter proteins on reconstituted transcription using purified nucleocapsids as templates. However, we have been able to show a dramatic stimulation of the polymerase activity in purified virions by the addition of purified C′ protein toin vitrotranscription reactions. Both the level and the fidelity of mRNA synthesis are stimulated by this protein. Evidence for the specificity of this effect comes from the fact that stimulation appears to be serotype specific; C′ protein of the Indiana serotype stimulates transcription by purified Indiana serotype virions but has a minimal effect on transcription by purified virions of the New Jersey serotype. We are continuing our studies to determine the mechanism of this stimulation.

Role of T helper cell precursor frequency on vesicular stomatitis virus neutralizing antibody responses in a T cell receptor beta chain transgenic mouse.

During most immune responses, T cells help antigen-specific B cells to make antibodies against the antigen. One of the contributions of T cells to antibody production is the induction of isotype switching from IgM to IgG, which is the most abundant isotype in blood serum during recall responses. Other features of memory responses are faster kinetics and higher titers of antibody in the serum. What causes a primary immune response to be different from a secondary is not yet very clear and, particularly, the influence of precursor frequencies of T and B cells on memory responses still remains to be answered. To address this issue, a transgenic (tg) mouse line (ADA) was developed; it expresses the beta chain (V beta 2) of a major histocompatibility complex class II-restricted T cell receptor (TcR) specific for the glycoprotein (G) of vesicular stomatitis virus (VSV) serotype Indiana (VSV-IND). These mice exhibit an increased precursor frequency of VSV-specific CD4+ T cells that leads to enhanced neutralizing IgG production against VSV in vivo in unprimed mice. The data indicate that increased frequency of naive specific helper T cells alone may account for features of a memory phenotype such as high titer of antibodies and isotype switching.

Replication and amplification of novel vesicular stomatitis virus minigenomes encoding viral structural proteins

We have developed a system in which vesicular stomatitis virus (VSV) minigenomes encoding viral structural proteins can be expressed from plasmids. These RNAs can be replicated, transcribed, and packaged into infectious particles when coexpressed with the other VSV proteins. The minigenomes contain either the glycoprotein (G protein) gene (GMG [stands for G minigenome]) or both the G and matrix (M) protein genes (GMMG [stands for G/M minigenome]) from the Indiana serotype of VSV flanked by the trailer and leader regions from the wild-type VSV genome. Northern (RNA) blot analysis showed that the minigenome RNAs were replicated and that a positive-sense replicative intermediate was synthesized when coexpressed with the nucleocapsid (N) protein and the two VSV polymerase proteins (phosphoprotein [P] and the large catalytic subunit [L]) in vivo. In addition, functional mRNAs were transcribed from the minigenome templates, and the appropriate encoded proteins were expressed. Expression of the G and M proteins from GMMG resulted in the assembly and release of infectious particles that could be passaged on cells expressing the N, P, and L proteins only. Amplification occurred during successive passages, and after four passages approximately 30% of the cells expressed both the G and M proteins. Analysis of the RNAs produced in the GMMG-infected cells also showed that the minigenomes accurately reproduced all of the replicative and transcriptional events that normally occur in a VSV-infected cell. GMMG is therefore a novel type of defective particle which encodes functional viral proteins critical to its own propagation.

Vesicular stomatitis virus Indiana glycoprotein as a T-cell-dependent and -independent antigen

The neutralizing immunoglobulin M (IgM) response to vesicular stomatitis virus (VSV) has been shown to be largely T-cell independent in several T-cell-deficient models of mice. By using different antigen froms of VSV, VSV antigen doses could be graded in vivo (infectious > > UV inactivated > formalin inactivated). The present study reveals a T-cell-dependent component of the neutralizing IgM response in nude mice given intravenous injections of low doses of noninfectious UV-inactivated VSV serotype Indiana (VSV-IND) only if the mice are transfused with VSV-IND-specific helper T cells. Instead, nude mice immunized with infectious VSV, which leads to greater antigen doses in vivo, were able to mount an IgM response in the absence of T cells. These results indicate that the IgM response to low doses of VSV-IND glycoprotein (G) is T-cell dependent. Nude mice immunized with infectious VSV also made a variable but low VSV-IND-neutralizing IgG response. A VSV-IND matrix (M)-specific helper T-cell line rendered this response more consistent, much higher, and longer lasting. Thus (i) VSV-G induces a mostly T-cell-independent but partially T-cell-dependent IgM (the latter can be visualized best at low doses of antigen) and (ii) the antibody response to VSV in nude mice proceeds through steps, i.e., IgM and IgG, that are dose dependent. The results suggest that the predominant role of helper T cells may be to expand and maintain the individual steps of differentiating B cells.

Common distribution of antigenic determinants and complementation activity on matrix proteins of two vesicular stomatitis virus serotypes.

To compare the antigenic and functional domains of the matrix (M) proteins of vesicular stomatitis virus (VSV) serotypes Indiana (VSV-Ind) and New Jersey (VSV-NJ), deletion mutants and chimeras were cloned in pBSM13 and expressed as in-frame lacZ fusion proteins in Escherichia coli. Non-cross-reactive monoclonal antibodies directed to the two antigenically distinct M proteins were tested by Western blot analysis to map three epitopes of VSV-Ind M protein and four epitopes of VSV-NJ M protein. Epitope 1 of the VSV-Ind M protein and epitope II of the VSV-NJ M protein both mapped to the highly basic N-terminal 34 amino acids of each homotypic M protein. Epitopes 2 and 3 of the VSV-Ind M protein and epitopes III and IV of the VSV-NJ M protein mapped to a region spanning amino acids 35 to 74. Epitope I of the VSV-NJ M protein mapped to a region between amino acid 75 and the C terminus. The similarity in location of the serotypically unique antigenic determinants of the two M proteins suggested that they may have a common functional domain. This hypothesis was substantiated by the finding that the two M proteins and various chimeras expressed in CV-1 cells by a recombinant vaccinia virus system were able to rescue M gene temperature-sensitive mutants of both VSV serotypes.

Characterization of T-helper epitopes of the glycoprotein of vesicular stomatitis virus.

The T-helper (Th) cell epitopes in the glycoprotein (GP) of vesicular stomatitis virus serotype Indiana (VSV-IND) were analyzed with a complete panel of overlapping synthetic peptides. Three Th-cell epitopes in C57BL/6 (H-2b) mice and two epitopes in BALB/c (H-2d) mice were defined by their ability to stimulate in vitro proliferation of virus-primed, CD8+ T-cell-depleted spleen cells in a class II-restricted manner. A series of CD4+, I-Ab-restricted T-cell hybridomas from VSV-primed C57BL/6 mice were characterized by their production of interleukin-2 and interleukin-3 upon stimulation with VSV-IND or purified VSV GP in vitro. Of nine hybridomas derived from three independent fusions, five were specific for amino acids (aa) 415 to 433 (p8) of VSV-IND GP, three recognized aa 52 to 71 (p41), and one reacted against aa 316 to 335 (p17). Fluorocytometric analysis of Th hybridomas or VSV-stimulated T-cell lines with monoclonal antibodies specific for the T-cell receptor V beta chain did not reveal obvious correlations between the T-cell receptor V beta gene segment used and the epitope recognized. All three peptides recognized by H-2b mice and both epitopes recognized by H-2d mice which were characterized in primed T-cell populations were capable of activating specific Th cells in vivo as measured by the induction of antibody class switch from immunoglobulin M (IgM) to IgG. Thus, the epitopes are relevant for VSV GP-specific Th response in vivo and are able to provide functional help for the production of anti-VSV-specific neutralizing IgG antibodies.

Localization of T Helper Cell Epitopes in the Vesicular Stomatitis Virus: The Nucleoprotein Is Responsible for Serotype Cross-Reactive T Help

The glycoprotein (G) of vesicular stomatitis virus (VSV) is known to contain the biologically relevant sites for virus-neutralizing antibodies as well as T helper (Th) cell epitopes. The capacity of other VSV proteins to elicit potent Th cell responses has not yet been investigated. Additionally, a short-lived cross-reactivity between the two serologically distinct VSV serotypes Indiana (IND) and New Jersey (NJ) on the T helper cell level has been reported. Here we address the question of whether the VSV nucleoprotein (N) or matrix protein (M) can elicit T help to VSV-G-specific B cells and which of the VSV proteins contains the elements responsible for the IND/NJ cross-reactivity. The N, G, and M of the VSV Indiana serotype produced in a recombinant baculovirus system were assayed for the ability to activate VSV-specific Th cells to induce immunoglobulin class switch of neutralizing antibody responses in vivo in C57BL/6 (H-2b) mice. All three VSV-IND proteins helped in the production of neutralizing IgG antibodies to the homologous VSV-Indiana serotype but only VSV-IND N was able to trigger an IgG response to the heterologous VSV-New Jersey serotype. This data suggest that Th epitope(s) in the VSV-IND N are responsible for the observed cross-reactivity of T helper cells.

The Influence of Antigen Organization on B Cell Responsiveness

The influence of antigen epitope density and order on B cell induction and antibody production was assessed with the glycoprotein of vesicular stomatitis virus serotype Indiana [VSV-G (IND)]. VSV-G (IND) can be found in a highly repetitive form the envelope of VSV-IND and in a poorly organized form on the surface of infected cells. In VSV-G (IND) transgenic mice, B cells were unresponsive to the poorly organized VSV-G (IND) present as self antigen but responded promptly to the same antigen presented in the highly organized form. Thus, antigen organization influences B cell tolerance.

Transcriptional activity and mutational analysis of recombinant vesicular stomatitis virus RNA polymerase.

The 241-kDa large (L) protein of vesicular stomatitis virus (VSV) is the multifunctional catalytic component of the viral RNA polymerase. A protocol has been developed for the synthesis of recombinant L protein that will support viral mRNA synthesis in vitro. COS cells were transfected with a transient expression vector (pSV-VSL1 [M. Schubert, G. G. Harmison, C. D. Richardson, and E. Meier, Proc. Natl. Acad. Sci. USA 82:7984-7988, 1985]) which contains the simian virus 40 late promoter for the transcription of a cDNA copy of the L protein of the Indiana serotype of VSV. Cytoplasmic extracts of these cells efficiently transcribed VSV mRNAs in vitro in conjunction with N protein-RNA template purified from virus and recombinant phosphoprotein synthesized in Escherichia coli. mRNA synthesis was completely dependent upon addition of both bacterial phosphoprotein and extracts from cells transfected with the L gene. Extracts from mock-transfected cells or from cells transfected with the expression vector alone did not support VSV RNA synthesis. RNA synthesis was proportional to the concentration of cell extract used, with an optimum of 0.2 mg/ml. Rhabdoviruses and paramyxoviruses contain a highly conserved GDNQ motif which was mutated in the transfected L gene. All constructs with mutations within the core GDN abrogated transcriptional activity except for the mutant containing GDD, which retained 25% activity. Conserved amino acid changes outside of the core GDN and changes corresponding to other paromyxovirus and rhabdovirus L proteins retained variable transcriptional activity. These findings provide experimental evidence that the GDN of negative-strand, nonsegmented RNA viruses is a variant of the GDD motif of plus-strand RNA viruses and of the XDD motif of DNA viruses and reverse transcriptases.

Formation of heterotrimers between the membrane-integrated and the soluble glycoproteins of vesicular stomatitis virus leads to their intracellular cotransport.

BHK cells infected with vesicular stomatitis virus serotype Indiana generate intracellularly two different types of glycoproteins: the authentic membrane-integrated G protein of virions and a smaller soluble Gs protein lacking the transmembrane and cytoplasmic domains which is secreted into the growth medium. A Gs1 protein species which is formed during or shortly after translation in the endoplasmic reticulum lumen is modified in the same way as the G1 protein by endoglycosidase H-sensitive oligosaccharides of the high-mannose type. Both G1 and Gs1 are almost simultaneously transported, trimmed, and processed into G2 and Gs2 species which possess carbohydrate side chains of the complex type, making both glycoproteins resistant to endoglycosidase H cleavage. Secretion of Gs2 protein into the growth medium and arrival of G2 protein on the cell surface occur concomitantly. Membrane-integrated G protein and the soluble Gs protein molecules oligomerize intracellularly into heterotrimers which can be immunoprecipitated after chemical cross-linking. Gs protein seems to contain sufficient structural information for the formation of heterotrimers which are efficiently transported to the cell surface. Heterotrimer formation between G and Gs proteins explains the rapid secretion of Gs molecules.

Antiviral protection by CD8+ versus CD4+ T cells. CD8+ T cells correlating with cytotoxic activity in vitro are more efficient in antivaccinia virus protection than CD4-dependent IL.

T cell-mediated protection against a recombinant vaccinia virus was evaluated in mice with respect to the relative contributions of CTL vs that of T cell-dependent IL and of CD4+ cells. H-2b mice primed with the wildtype of vesicular stomatitis virus serotype Indiana (VSV-IND wt) mount an in vitro measurable cytotoxic response against the nucleoprotein (NP) of VSV-IND and are protected against a challenge infection with a vaccinia-VSV recombinant virus expressing the NP of VSV-IND (vacc-IND-NP). Their protective mechanism was highly susceptible to in vivo depletion of CD8+ T cells, but resistant to CD4+ depletion or treatment with anti-IFN-gamma and anti-TNF-alpha. Surprisingly, also VSV-CTL nonresponder H-2k mice were protected against a challenging infection with vacc-IND-NP when primed with VSV-IND wt. In contrast to the CTL responder H-2b mice, this protection was highly susceptible to CD4+ T cell depletion and to anti-IFN-gamma or anti-TNF-alpha treatment, but resistant to CD8+ T cell depletion. Antibodies were not responsible because they failed to transfer protection; in contrast CD4+ T cells conferred significant protection. VSV-CTL responder H-2b and nonresponder H-2k mice were protected almost equally well against a challenge dose of 10(3) pfu vacc-IND-NP inoculated intracerebrally. However, after intracerebral challenge with 5 x 10(6) pfu vacc-IND-NP, the CTL nonresponder mice died, whereas the CTL responder mice eliminated the virus by day 5. These results collectively show that CD4+ T cell-dependent IL may mediate antiviral protection, but their efficiency is relatively weak compared with CD8-mediated protection correlating with cytotoxic activity in vitro.

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.

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 bydeletion 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-N1 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 epitopespecific monoclonal antibodies.

L929 cells infected with temperature sensitive mutants of vesicular stomatitis virus: virus replication is necessary for induction of changes in membrane permeability.

Infection of L929 murine cells with vesicular stomatitis virus (VSV) results in inhibition of host protein synthesis and appearance of membrane alterations at a time when cells are still actively engaged in viral protein synthesis. VSV temperature-sensitive (ts) mutants have been used to explore the role(s) played by the virus-coded proteins in the genesis of these effects. Cells were infected with each of five ts mutants representing the known complementation groups of VSV Indiana serotype, and incubated at permissive (32 degrees C) and non-permissive temperatures (39 degrees C). Protein synthesis in the presence and absence of Hygromycin B (Hyg. B) was analyzed during virus infection via incorporation of 35S-methionine in acid-precipitable material and SDS-polyacrylamide gel electrophoresis. Data indicate that mutants belonging to groups I (L protein), II (NS protein) and IV (N protein) do not inhibit host protein synthesis and do not induce any membrane changes when grown at the non-permissive temperature. Mutants of group III (M protein) and V (G protein), instead, do inhibit cell protein synthesis and induce membrane changes also when grown at the non-permissive temperature; this suggests that these effects do not correlate with the biological activity of these proteins and their interaction with the cellular membrane. On the other hand, mutants exhibiting defective steps of nucleocapsid replication are apparently unable to induce these effects once more suggesting that virus replication per se is essential, as also indirectly shown by experiments employing cycloheximide to mimic shut-off.

A novel approach for the production of monoclonal antibodies using infectious vaccinia virus recombinants.

We describe a novel approach of producing monoclonal antibodies (MABs) to one specific protein of a virus or other agent consisting of several proteins, without the use of purified antigen in either the immunization or screening phase of the procedure. This method has general application in the production of MABs when the antigen cannot be obtained in a pure form, but the gene is available. We illustrate this application by producing MAB specific to the nucleocapsid protein (N) of vesicular stomatitis virus serotype Indiana (VSV-IN) from BALB/c mice immunized with an infectious vaccinia virus recombinant vector (v38) that expresses the N gene of VSV-IN. This novel method of immunization obviates the need for initial purification of the protein antigen and injection of adjuvants with the isolated protein as is done in traditional MAB production.

The soluble glycoprotein of vesicular stomatitis virus is formed during or shortly after the translation process.

Gs protein is a shorter, soluble form of the viral G protein of vesicular stomatitis virus (VSV) lacking the membrane-anchoring domain. Production of Gs protein appears to be a general property of VSV because infection of BHK-21 cells by five different isolates of the VSV serotype Indiana led in all cases to the synthesis of Gs protein. Moreover, it is formed in a variety of eucaryotic cell lines after VSV infection. In pulse-chase experiments, we observed a time-dependent change in the ratio of G to Gs protein released into the growth medium, suggesting that Gs is formed intracellularly rather than on the cell surface. Further experiments revealed that Gs protein can be synthesized in vitro in the reticulocyte lysate system after addition of a viral mRNA fraction and in a coupled transcription-translation system with VSV core particles. In the presence of microsomal membranes both G and Gs protein were glycosylated in the reticulocyte lysate, confirming that the authentic Gs protein is synthesized in vitro. The addition of various protease inhibitors to the cell-free system and variation of the incubation conditions did not alter the ratio of G to Gs formation. Taken together, these experiments suggest strongly that Gs protein is not a product of a membrane-associated proteolytic activity but is formed during or shortly after the translation process. Our attempts to detect a specific, shorter mRNA coding for the Gs protein by molecular hybridization procedures did not reveal the existence of such a mRNA species.

Protection against lethal viral infection by neutralizing and nonneutralizing monoclonal antibodies: distinct mechanisms of action in vivo.

Monoclonal antibodies (MAb) reactive with the glycoprotein of vesicular stomatitis virus (VSV) serotypes Indiana (VSV-Ind) and New Jersey (VSV-NJ) were used to protect mice against lethal infection. MAb which reacted with a number of distinct epitopes and which could neutralize the virus in vitro could also protect against infection in vivo. MAb which could not neutralize the virus in vitro but which were specific for the glycoprotein of a single serotype were also able to protect mice against lethal VSV challenge. Interestingly, a group of MAb which cross-reacted with the glycoproteins of VSV-Ind and VSV-NJ could passively protect against challenge with either serotype. It was shown that as early as 2 h after infection, neither neutralizing nor nonneutralizing MAb could protect. Nonneutralizing MAb were found to be less effective at in vivo protection than neutralizing MAb. Furthermore, nonneutralizing MAb demonstrated a much lower binding efficiency to intact virions than did neutralizing MAb. These observations, plus the fact that the nonneutralizing MAb could lyse virus-infected cells in the presence of complement, suggested that in vivo protection by these antibodies may involve cell-associated viral determinants. To compare the mechanisms by which neutralizing and nonneutralizing MAb protected in vivo, F(ab')2 fragments were used in protection experiments. Although the F(ab')2 of a neutralizing MAb was still able to protect animals lethal virus challenge, the F(ab')2 of a cross-reactive nonneutralizing MAb was unable to do so. The reactivity of nonneutralizing MAb with virions and the apparent necessity of an intact Fc portion for protection further distinguish these antibodies from those MAb that are able to neutralize VSV solely by binding to the glycoprotein.

Unusual heterogeneity in the glycosylation of the G protein of the Hazelhurst strain of vesicular stomatitis virus

The asparagine-linked oligosaccharides of the G protein of the Hazelhurst subtype of the New Jersey serotype of vesicular stomatitis virus (VSV) have been compared with the oligosaccharides from the G protein of the well-characterized Indiana serotype of VSV, with baby hamster kidney cells in monolayer culture as the host for both viruses. [ 3H]Glucosamine- and [ 3H]mannose-labeled glycopeptides from the G protein of purified virus were analyzed by the combined techniques of endo-β- N-acetylglucosaminidase H (ENDO-H) digestion, concanavalin A and lentil lectin affinity chromatography, and Bio-Gel P-4 chromatography. Although almost all of the Indiana G protein oligosaccharides were acidic-type structures, as expected from previous studies; the Hazelhurst G protein contained a mixture of acidic-type, hybrid-type containing sialic acid, and neutral-type (predominantly Man 5–6GlcNAc 2-Asn) structures. The vast majority of acidic-type oligosaccharides from both the Hazelhurst and Indiana G proteins were diantennary structures, with less than half containing fucose linked to the innermost N-acetylglucosamine. Additional analysis of the Hazelhurst G protein by ENDO-H digestion and gel electrophoresis suggested that some of the mature G polypeptides contained acidic-type structures at both glycosylation sites, whereas the remainder contained an ENDO-H-resistant, acidic-type structure at one site and an ENDO-H-sensitive, hybrid- or neutral-type structure at the other site.

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.