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.

FIG. 1.

Construction and initial characterization of an inducible I7L mutant. (A) Diagram of the genome of vI7Li. Important features include the presence of the bacteriophage T7 RNA polymerase gene (T7 pol) regulated by the VV late P11 promoter (PL) and the E. coli lac operator (lacO); the E. coli lac repressor (lacI) regulated by the VV early-late P7.5 promoter (PE/L); the GFP gene regulated by the P11 promoter and replacing a large segment of the original I7L gene; and a new copy of the I7L gene (I7L.2) regulated by the bacteriophage T7 promoter (PT7), the lac operator, and the encephalomyocarditis (EMC) leader. TK_L, TK_R, HA_L, and HA_R represent left and right flanking segments of the TK and HA genes. gpt, E. coli guanine phosphoribosyltransferase gene. (B) Effect of IPTG on plaque formation. BS-C-1 cell monolayers were infected with the parental virus vT7LacOI, the intermediate virus vI7L/I7Li, or vI7Li in the presence or absence of 20 μM IPTG. After 48 h, plaques were visualized by staining with crystal violet. (C) Microscopic visualization of cells infected with vI7Li in the presence or absence of IPTG and expressing GFP.

FIG. 2.

Effect of IPTG on production of infectious vI7Li. (A) BS-C-1 cells were infected with vT7LacOI (•), vI7L/I7Li (▪), or vI7Li (▴) at a multiplicity of infection of 5 and incubated in the presence of 0 to 250 μM IPTG. Twenty-four hours after infection, virus titers in the presence of 20 μM IPTG were determined by plaque assays. (B) BS-C-1 cells were infected with vT7LacOI (•) or with vI7Li in the presence (▪) or absence (▴) of 20 μM IPTG. Cells were harvested at the indicated times after infection, and virus titers were determined as described above.

FIG. 3.

Electron microscopy of cells infected with vI7Li in the absence of IPTG. BS-C-1 cells were infected with vI7Li at a multiplicity of infection of 5. Twenty-four hours after infection, cells were fixed and embedded in Epon, and ultrathin sections were prepared for transmission electron microscopy. (A) Clusters of IV, some with nucleoids (n), are shown. In addition, there are dense particles, many of which are irregularly shaped (arrows). (B) Cluster of intracellular enveloped virions and one cell-associated extracellular enveloped virion (on the right). The arrows marked “c” point to poorly formed cores.

FIG. 4.

Expression and processing of viral proteins. (A) Effect of IPTG on the expression of I7 protein. Cells were left uninfected (UN) or were infected with vI7Li in the presence of 0 to 250 μM IPTG or with wild-type VV (WR) at a multiplicity of infection of 5. The cells were harvested after 18 h, and the proteins in the cellular extracts were separated by SDS-PAGE and detected by Western blotting with a polyclonal antibody to I7 (α-I7). The apparent molecular mass, in kilodaltons, is indicated on the right. (B) Effect of IPTG on processing of A17 protein. BS-C-1 cells were infected and analyzed as described for panel A, except that Western blotting was performed with a polyclonal antibody to an N-terminal peptide of the mature A17 protein (α-A17). The apparent masses of precursor and product proteins are shown on the right. (C) Effect of temperature on processing of A17 protein expressed by VV ts16 mutant. BS-C-1 cells were left uninfected or infected with WR or the ts16 mutant and were maintained at the permissive temperature of 31°C or the nonpermissive temperature of 39°C. The cells were analyzed as described for panel B. (D) Effect of rifampin on processing of A3 and A17 proteins. BS-C-1 cells were infected with WR in the absence (−) or presence (+) of 100 μg of rifampin per ml and were analyzed as described for the other panels, except for the use of a polyclonal antibody to the A3 (α-A3) or A17 protein. The apparent masses of the precursor and product proteins are shown on the right.

FIG. 5.

The putative catalytic site of I7 is required for cleavage of the A17 membrane protein and the L4 core protein. (A) I7 requirement for cleavage of A17. Cells were infected with vI7Li at a multiplicity of infection of 3 in the absence of IPTG and transfected with the indicated vector plasmid, a plasmid expressing A17, or a wild type (wt) or mutated (C328S or H241R) form of I7 regulated by the VV late P11 promoter and containing a C-terminal influenza virus HA epitope tag. Eighteen hours after infection, cells were harvested and proteins were separated by SDS-PAGE and detected by Western blotting with an antibody to the N-terminal peptide of the mature A17 protein (α-A17). The apparent molecular masses of the precursor and cleaved forms of A17 are shown on the right. (B) I7 requirement for cleavage of L4. Infection and transfection were performed as described above, with an additional plasmid that expressed L4 regulated by a synthetic late promoter and containing an influenza virus HA epitope tag. The Western blot was analyzed with MAb HA.11 to the HA epitope tag. The apparent molecular masses of the I7 protein (46 kDa) and the precursor (28 kDa) and cleaved (25 kDa) forms of the L4 protein are shown on the right.

FIG. 6.

Processing of mutated A17 proteins containing an internal V5 tag. (A) Diagram of A17 substrate showing the location of the internal V5 tag and the sequence around the three glycines that were mutated to alanine. (B) Analysis of A17 cleavage. Cells were infected with vI7Li at a multiplicity of infection of 3 in the absence of IPTG and were transfected with a vector plasmid or a plasmid expressing A17 with an internal V5 tag containing no other mutations (wt) or a mutation by which a glycine(s) was replaced with an alanine(s). Eighteen hours after infection, cells were harvested and proteins were separated by SDS-PAGE in a Tris-glycine-4 to 20% polyacrylamide gel and detected by Western blotting with a MAb to the V5 tag. The apparent molecular masses of the precursor and cleaved A17 are shown on the right.

FIG. 7.

Processing of mutated A17 proteins containing an N-terminal V5 tag and a C-terminal HA tag. (A) Diagram of A17 showing the locations of the V5 and HA tags and the sequences around the three glycines that were mutated to alanine. (B) Analysis of A17 cleavage. Cells were infected with vI7Li at a multiplicity of infection of 3 in the absence of IPTG and were transfected with a vector plasmid or a plasmid expressing A17 with an N-terminal V5 tag and a C-terminal HA tag and containing no other mutations (wt) or a mutation by which the indicated glycine(s) was replaced with an alanine(s). Eighteen hours after infection, cells were harvested and proteins were separated by SDS-PAGE in a Tris-glycine-4 to 20% polyacrylamide gel (upper panel) or a Tricine-10% polyacrylamide gel (lower panel) and detected by Western blotting with a MAb to the V5 tag (α-V5) or the HA tag (α-HA). The apparent molecular masses of the precursor and cleaved A17 are shown on the right.

FIG. 8.

Processing of mutated L4 proteins. (A) Diagram of L4 showing the C-terminal location of the HA epitope tag and the sequences around the two glycines that were mutated to alanine. (B) Analysis of L4 cleavage. Cells were infected with vI7Li at a multiplicity of infection of 3 in the absence of IPTG and were transfected with a vector plasmid, a plasmid expressing wild-type I7, or a plasmid expressing L4 with a C-terminal HA tag and containing no other mutations (wt) or a mutation by which the indicated glycine(s) was replaced with an alanine(s). Eighteen hours after infection, cells were harvested and proteins were separated by SDS-PAGE and detected by Western blotting with a MAb to the HA epitope tag (HA.11). The apparent molecular masses of I7 (46 kDa) and the precursor and cleaved forms of L4 (28 and 25 kDa, respectively) are shown on the right.