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. 2006 Jul;80(14):7159–7168. doi: 10.1128/JVI.00592-06

FIG. 2.

FIG. 2.

Us2 interacts with ERK in PRV-infected cells and directs ERK incorporation into virions. (A) Total ERK (T-ERK) was immunoprecipitated (IP) from NIH 3T3 cells infected with PRV174 (Us2-null mutant) or wt Becker and analyzed by Western blotting using antiserum specific for Us2 (top) and gB (bottom). The immunoglobulin heavy chain (IgH) from the immunoprecipitation cross-reacted with the secondary antiserum used to detect glycoprotein gB. PRV gB is a type I membrane protein that is cleaved in the Golgi from the preprocessed monomer pgB into two smaller subunits that are linked by disulfide bonds. These subunits have molecular masses of 69 kDa and 58 kDa and represent the amino-terminal and carboxy-terminal “halves” of the molecule, respectively (gB). Positive control (+ control) is an extract prepared from PRV Becker-infected cells. The asterisk denotes nonspecific bands that cross-react with the Us2 antiserum. (B) Phosphorylated ERK (P-ERK) was immunoprecipitated (IP) from NIH 3T3 cells infected with PRV174 or Becker and analyzed by Western blotting using antiserum specific for Us2 (top) and gB (bottom). The asterisk denotes nonspecific bands that cross-react with the Us2 antiserum. Positive control (+ control) is an extract prepared from PRV Becker-infected cells. IgH, immunoglobulin heavy chain. (C) Purified virions from Becker and PRV174-infected cell supernatants were isolated as described in Materials and Methods. Treatment of virions with proteinase K in the absence of SDS degraded any proteins not protected by the virion envelope. Proteins found in the tegument are protected by the virion envelope. gB was used as a virus loading control and as a control for the activity of the protease in the absence of SDS. Western blot analysis was performed using antiserum specific for total ERK (top), Us2 (middle), or gB (bottom). +, with; −, without. (D) A PK15 cell line that stably expresses Us2 was infected with PRV174. Virions were purified from infected cell supernatants and treated with proteinase K and/or SDS. Note that the incorporation of Us2 and ERK into the tegument is restored. +, with; −, without.