Role of the I7 Protein in Proteolytic Processing of Vaccinia Virus Membrane and Core Components

Abstract

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.Vaccinia virus (VV), the best-studied member of the Poxviridae, is a complex enveloped DNA virus that replicates in the cytoplasm of infected cells (15). Viral DNA replication, transcription, the accumulation of viral structural proteins, and the assembly of virions occur in discrete juxtanuclear viral factories. Morphogenesis begins with the formation of crescentshaped membranes that engulf dense viroplasm to form spherical immature virions (IV) (6). Maturation of the IV into infectious intracellular mature virions (IMV) involves a poorly understood process in which at least three core proteins, namely P4a, P4b, and P25, encoded by the A10L, A3L, and L4R open reading frames (ORFs), respectively, undergo proteolytic processing (12,16,26) at a consensus AG/X motif (21-23, 28). Initial evidence that processing is coupled to virus maturation was obtained by using the drug rifampin, which reversibly blocks an early step in virus assembly and the cleavage of core proteins (12, 17). Subsequently, numerous temperature-sensitive (ts) mutants were shown to have defects in virus assembly and the processing of core proteins under nonpermissive conditions. One IMV membrane protein, encoded by the A17L ORF, also undergoes proteolytic cleavages at AG/X sites (2,18,21,28). The processing of membrane and core proteins appears to be separable, as two studies described no effect of rifampin on the cleavage of A17 (2, 18) but a third study concluded that rifampin blocked cleavage of this protein (28...

Activity, Specificity, and Probe Design for the Smallpox Virus Protease K7L

“…K7L orthologs are present and presumably required in all orthopoxviruses. By employing transient expression and conditional lethal virus analyses, it has previously been demonstrated that I7L, the vaccinia ortholog of K7L, is required for core protein processing (7,16,18). A seminal study used extracts of virally infected cells containing I7L to demonstrate that the enzyme is a cysteine protease able to cleave core protein precursors P4a, P4b, and P25K (17).…”

“…Furthermore, we found that polyanions have differential effects on the activity of the enzyme on protein vis à vis peptide substrates. Together, these characteristics point to a regulation of K7L that is multivariate and more complex than for other Clan CE members, possibly as a consequence of the stringent requirement for its activity to be localized predominantly or exclusively to the condensing virion (7,16,18).…”

“…The gene products of K7L (variola) and I7L (vaccinia) are 423-residue proteases that belong to the MEROPS Clan CE (7,15,16). The two proteases differ at only four residues and are thus likely to be functionally very similar.…”

“…Residues important in catalysis of other members of Clan CE are conserved in K7L/I7L and suggest that it is a cysteine protease (confirmed experimentally by Byrd and Hruby group (17)). Based on in vivo cleavage data, I7L processes one envelope protein (P21K (A17L)) and four core proteins (P4a (A10L), P4b (A3L), P17K (A12L), and P25K (L4R)) (7,16,18,19) at sites with the AG-X motif (where X is generally a small residue). AG-X motifs occur in other vaccinia proteins as well, but they are not processed, suggesting that additional substrate motifs are required for recognition (12).…”

Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space

“…Recently, other proteinases have been added to the AVP family. Among viruses, this includes vaccinia virus (41) and African swine fever virus (42). Chlamydia trachomatis has a gene similar to that of AVP (43).…”