DELETION OF THE VACCINIA VIRUS B5R-GENE ENCODING A 42-KILODALTON MEMBRANE GLYCOPROTEIN INHIBITS EXTRACELLULAR VIRUS ENVELOPE FORMATION AND DISSEMINATION

The structure, formation, and function of the virion membranes are amo ng the least well understood aspects of vaccinia virus replication. In this study, we investigated the role of gp42, a glycoprotein componen t of the extracellular enveloped form of vaccinia virus (EEV) encoded by the B5R gene. The B5R gene was deleted by homologous recombination from vaccinia virus strains IHD-J and WR, which produce high and low l evels of EEV, respectively. Isolation of recombinant viruses was facil itated by the insertion into the genome of a cassette containing the E scherichia coli gpt and lacZ genes flanked by the ends of the B5R gene to provide simultaneous antibiotic selection and color screening. Del etion mutant viruses of both strains formed tiny plaques, and those of the IHD-J mutant lacked the characteristic comet shape caused by rele ase of EEV. Nevertheless, similar yields of intracellular infectious v irus were obtained whether cells were infected with the B5R deletion m utants or their parental strains. In the case of IHD-J, however, this deletion severely reduced the amount of infectious extracellular virus . Metabolic labeling studies demonstrated that the low extracellular i nfectivity corresponded with a decrease in EEV particles in the medium . Electron microscopic examination revealed that mature intracellular naked virions (INV) were present in cells infected with mutant virus, but neither membrane-wrapped INV nor significant amounts of plasma mem brane-associated virus were observed. Syncytium formation, which occur s in cells infected with wild-type WR and IHD-J virus after brief low- pH treatment, did not occur in cells infected with the B5R deletion mu tants. By contrast, syncytium formation induced by antibody to the vir al hemagglutinin occurred, suggesting that different mechanisms are in volved. When assayed by intracranial injection into weanling mice, bot h IHD-J and WR mutant viruses were found to be significantly attenuate d. These findings demonstrate that the 42-kDa glycoprotein of the EEV is required for efficient membrane enwrapment of INV, externalization of the virus, and transmission and that gp42 contributes to viral viru lence in strains producing both low and high levels of EEV.