Vaccinia virus G1 protein, a predicted metalloprotease, is essential for morphogenesis of infectious virions but not for cleavage of major core proteins.
Genes encoding orthologs of the vaccinia virus G1 protein are present in all poxviruses for which sequence information is available, yet neither the role of the protein nor its requirement for virus replication is known. G1 was predicted to be involved in the cleavage of core proteins, based on a transfection study and the presence of an HXXEH motif found in a subset of metallopeptidases. In the present study, we engineered a recombinant vaccinia virus containing a single copy of the G1L gene with a C-terminal epitope tag that is stringently regulated by the Escherichia coli lac repressor. In the absence of inducer, expression of G1 was repressed and virus replication was inhibited. Rescue of infectious virus was achieved by expression of wild-type G1 in trans, but not when the putative protease active site residues histidine-41, glutamate-44, or histidine-45 were mutated. Nevertheless, the synthesis and proteolytic processing of major core and membrane proteins appeared unaffected under nonpermissive conditions, distinguishing the phenotype of the G1L mutant from one in which the gene encoding the I7 protease was repressed. Noninfectious virus particles, assembled in the absence of inducer, did not attain the oval shape or characteristic core structure of mature virions. The polypeptide composition of these particles, however, closely resembled that of wild-type virus. Full-length and shorter forms of the G1 protein were found in the core fraction of virus particles assembled in the presence of inducer, suggesting that G1 is processed by self-cleavage or by another protease.
Ansarah-Sobrinho
Camilo
C
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institues of Health, Bethesda, MD 20892-0445, USA.
Moss
Bernard
B
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D020043:Genes, Essential; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D045726:Metalloproteases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009024:Morphogenesis; D011995:Recombination, Genetic; D016415:Sequence Alignment; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity; D014758:Viral Core Proteins / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000254:growth & development* / Q000378:metabolism
D014758:Viral Core Proteins; D014764:Viral Proteins; D045726:Metalloproteases