Molecular dissection of the vaccinia virus I7L core protein proteinase

Abstract

The vaccinia virus I7L gene product is predicted to be a cysteine proteinase and is demonstrated in this study to be responsible for cleavage of each of the three major core protein precursors (P4a, P4b, and P25K) in vivo. Mutagenesis of the putative catalytic triad of I7L or of the cleavage sites in the core protein precursors inhibits processing. A truncated protein lost the ability to cleave the core protein precursors.

FIG. 1.

Characterization of I7L and predicted hydrophobicity plot of the I7L protein made with the Kyte-Doolittle program. The positions of seven highly conserved amino acids, including the putative catalytic triad, and the position of the ts16 mutation are indicated by arrows. The positions of the amino acids are indicated on the x axis. Sequence similarity to the corresponding gene in variola virus, camelpox virus, and monkeypox virus is indicated by rectangles underneath the plot, with differing amino acids indicated at the correct positions by black bars. Sequence similarity of the conserved catalytic domain between VV I7L, the ASFV protease, adenovirus protease (ADE2), and a yeast cysteine protease (Ulp1) is indicated at the bottom of the figure, with arrows pointing at the highly conserved amino acids.

FIG. 2.

VV core protein cleavage sites. Schematic representation of the three major core protein precursors (P4a, P4b, and P25K) along with full-length and truncated I7L. The positions of the AGX cleavage sites are indicated by the amino acid number of the glycine.

FIG. 3.

Proteolytic processing of the core protein precursors. BSC₄₀ cells were infected with VV ts16 and transfected with plasmids containing either I7L, P4a, P4b, or a mixture of these. Cells were harvested 24 h postinfection, and the extracts were analyzed by Western blot with anti-Flag antisera. (A) Processing of P4b. In each lane, cells are infected with ts16 and then transfected with either substrate alone or with substrate plus enzyme. The substrate is pP4b or pP4bIDI (where the AGA site is mutated to an IDI), and the enzyme is either pI7L or pH241A (pI7L with His 241 mutated to Ala). (B) Processing of P4a. In each lane, cells are infected with ts16 and then transfected with either substrate alone or with substrate plus enzyme. In this case, the substrate is either pP4a, pP4aIDI696 (P4a with the AGT site mutated to an IDI), or pP4aIDI613 (P4a with the AGS site mutated to an IDI).

FIG. 4.

Ability of mutant I7L enzymes to cleave the core protein precursors. BSC₄₀ cells were infected with VV ts16 and transfected with plasmids containing either P4a, P4b, or P25K and cotransfected with either pI7L or one of the seven mutant I7L plasmids. Cells were harvested 24 h postinfection, and the extracts were analyzed by Western blot with anti-Flag antisera to determine cleavage of the precursor protein. The top and middle panels show P25K and P4b, respectively, transfected with each mutant I7L plasmid, and the bottom panel shows P4a transfected with each mutant enzyme.