FIG. 2.

Identification of the 42-kDa protein as the product of the G7L ORF. (A) Affinity purification of the A30L protein complex. Proteins extracted from cells infected with vA30LiHA in the presence of IPTG were purified using a high-affinity anti-HA affinity matrix. The F.T (Flowthrough) lane contains material that did not bind to the HA matrix. The HA matrix was washed with lysis buffer (wash lanes, fractions 1 to 3), and the proteins were eluted with the HA peptide (HApep lanes, fractions 1 to 3) followed by glycine (glycine lanes, fractions 1 to 4). Proteins from each fraction were analyzed by electrophoresis on an SDS-10 to 20% polyacrylamide gel in Tricine buffer followed by silver staining. Bands corresponding to the A30L and 42-kDa proteins are indicated. Numbers on the left indicate the positions and molecular masses in kilodaltons of marker proteins. (B) Predicted amino acid sequence of the VV G7L ORF. Peptides identified by MS are underlined. Amino acids comprising the peptide used to produce polyclonal antibody are in italics, and consensus proteolysis cleavage sites are indicated by arrows. (C) Reactivity of the 42-kDa protein with G7L protein antiserum. (Left) Lysates of uninfected (lane U) and VV WR-infected BS-C-1 cells were resolved by SDS-PAGE, reacted with G7L peptide antiserum by Western blotting, and detected by chemiluminescence. (Right) Extracts of cells infected with the vA30LiHA virus in the presence (+) or absence (−) of 100 μM IPTG were incubated with the high-affinity anti-HA affinity matrix, and bound proteins were analyzed by Western blotting with G7L protein antiserum. The positions of migrations and molecular masses of marker proteins are indicated on the left.