Role of the I7 protein in proteolytic processing of vaccinia virus membrane and core components.
Certain core and membrane proteins of vaccinia virus undergo proteolytic cleavage at consensus AG/X sites. The processing of core proteins is coupled to morphogenesis and is inhibited by the drug rifampin, whereas processing of the A17 membrane protein occurs at an earlier stage of assembly and is unaffected by the drug. A temperature-sensitive mutant with a lesion in the I7L gene exhibits blocks in morphogenesis and in cleavage of core proteins. We found that the mutant also failed to cleave the A17 membrane protein. To further investigate the role of the putative I7 protease, we constructed a conditional lethal mutant in which the I7L gene was regulated by the Escherichia coli lac repressor. In the absence of an inducer, the synthesis of I7 was repressed, proteolytic processing of the A17 membrane protein and the L4 core protein was inhibited, and virus morphogenesis was blocked. Under these conditions, expression of the wild-type I7 protein in trans restored protein processing. In contrast, rescue did not occur when the putative protease active site residue histidine 241 or cysteine 328 of I7 was converted to alanine. The mutation of an authentic AG/A and an alternative AG/S motif of L4 prevented substrate cleavage. Similarly, when AG/X sites of A17 were mutated, I7-induced cleavages at the N and C termini failed to occur. In conclusion, we provide evidence that I7 is a viral protease that is required for AG/X-specific cleavages of viral membrane and core proteins, which occur at early and late stages of virus assembly, respectively.
Ansarah-Sobrinho
Camilo
C
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 4 Center Drive, MSC 0445, Bethesda, MD 20892-0445, USA.
Moss
Bernard
B
D002460:Cell Line; D010450:Endopeptidases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism*; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly; D014779:Virus Replication
C081257:A17L protein, Vaccinia virus; D008565:Membrane Proteins; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; C080700:protein I7, vaccinia virus; D010450:Endopeptidases