Ruth M. Snyder

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.

Spontaneous mutations leading to antigenic variations in the glycoproteins of vesicular stomatitis virus field isolates.

Strains of vesicular stomatitis virus, New Jersey serotype (VSV-NJ), isolated from diseased cattle or swine were examined by genomic RNA sequencing for genetic diversity potentially leading to antigenic variations in their type-specific glycoproteins as determined by reactivity with epitope-specific monoclonal antibodies (MAbs). Seven field isolates recovered in Colorado, New Mexico, Georgia, and Mexico during the widespread 1982-1985 epizootic in the western United States resembled the prototypic 1952 Hazelhurst subtype by partial sequence homology, but amino acid reversions to the 1949 Ogden subtype occurred frequently. When studies were performed with MAbs directed to the Ogden subtype glycoprotein, relatively limited antigenic variation, and only in neutralization epitope VIII, was noted among two of five epizootic isolates from Colorado and New Mexico. However, amino acid differences in the glycoprotein of a 1983 isolate from an enzootic region of Georgia resulted in major antigenic deficiencies in epitopes V, VI, and VII as determined by Western blotting and neutralization of infectivity with epitope-specific MAbs. Quite a few genetic but no antigenic differences were noted in an enzootic 1984 isolate from Mexico, a potential origin of the United States epizootic. Marked or complete loss of epitopes VII, VI, VIII, and V can be traced to spontaneous mutations leading to amino acid substitutions at glycoprotein positions 199, 263, 275, and 317, respectively, in the enzootic Georgia isolate 07/83-GA-P and the epizootic New Mexico isolate 06/85-NM-B. By comparison, closely adjacent amino acid substitutions at glycoprotein positions 210, 268, 277, and 364 occurred in epitope-deficient mutants selected for resistance to neutralization by MAbs specific for epitopes VII, VI, VIII, and V, respectively. Two neutralization epitopes designated X and XI were found to be unique for the G protein of the 1952 Hazelhurst isolate..../52-GA-P. The epitope X-specific MAb H21, in particular, failed to neutralize the infectivity not only of the Ogden subtype..../49-UT-B but also was ineffective against all the 1982-1985 field isolates. The classical 1952 Hazelhurst strain of VSV-NJ is genetically and antigenically quite different from those viruses isolated during the 1982-1985 epizootic.

Monoclonal antibodies to the matrix protein of vesicular stomatitis virus (New Jersey serotype) and their effects on viral transcription.

Of 33 hybridomas raised by immunization of BALB/c mice with the matrix (M) protein of the New Jersey serotype of vesicular stomatitis virus (VSV), 17 secreted monoclonal antibodies (mAb) of the IgG isotype and, unexpectedly, 16 of the IgM isotype. All these monoclonal antibodies bound strongly to VSV-New Jersey M protein by ELISA, immunoprecipitation, and immunoblotting assays, but exhibited only slight or no cross-reactivity with the M protein of VSV-Indiana. Four antigenic determinants of VSV-New Jersey M protein could be identified by competitive binding of 125I-labeled monoclonal antibodies but three of these epitopes exhibited partial overlap. Monoclonal antibodies to two epitopes reversed the inhibitory effect of M protein on in vitro transcription of VSV-New Jersey ribonucleoprotein. However, monoclonal antibodies to the other two epitopes had little effect on M-protein transcription inhibition but actually increased significantly the transcriptional inhibitory effect of M protein under certain experimental conditions. Monoclonal antibodies to all four epitopes reacted strongly with the M protein of the tsC1 mutant of VSV-New Jersey which is restricted in transcription inhibition.

Regulation of viral transcription by the matrix protein of vesicular stomatitis virus probed by monoclonal antibodies and temperature-sensitive mutants

The ability of the matrix (M) protein of wild-type vesicular stomatitis virus (VSV) to regulate viral transcription was studied with monoclonal antibodies and temperature-sensitive (ts) mutants in complementation group III, the M proteins of which are restricted in transcription inhibition. The marked inhibition of transcription by VSV ribonucleoprotein (RNP) cores complexed with M protein (RNP/M) was reversed by antibody to epitope 1. Antibodies to epitopes 2 and 3 not only failed to reverse the transcription-inhibitory activity of isolated M protein but actually increased M-protein inhibition of transcription in a reconstituted system. Monoclonal antibodies to epitopes 2 and 3 strongly bound to M proteins from all wild-type and ts-mutant virions, but monoclonal antibody to epitope 1 completely failed to bind to the M protein of ts023(III) even though it reacted strongly with M proteins of mutants tsG31(III) and tsG33(III). The M protein of a tsO23 revertant (R11) completely recovered its capacity to inhibit transcription and to bind monoclonal antibody to epitope 1, whereas the M proteins of three other revertants remained restricted in their capacity to inhibit transcription and to bind monoclonal antibody to epitope 1. These studies indicate that exposure of epitope 1 on the surface of M protein is essential for inhibiting transcription by VSV RNP cores.