Role of the 17 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 17L 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 17 protease, we constructed a conditional lethal mutant in which the 17L gene was regulated by the Escherichia coli lac repressor. In the absence of an inducer, the synthesis of 17 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 17 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 17 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, 17-induced cleavages at the N and C termini failed to occur. In conclusion, we provide evidence that 17 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.