Fig 9.

Us2 is a ubiquitin-interacting protein. (A and B) Vero cells were infected with WT HSV-2, HSV-2 ΔUs2, or recombinant HSV-2 with Flag-tagged Us2 (A) or with WT HSV-1 or recombinant HSV-1 with Flag-tagged Us2 (B). At 6 hpi, cell lysates were prepared, and proteins were immunoprecipitated (IP) using an anti-Flag affinity gel. Immunoprecipitated proteins were detected using antibodies against HSV-1 Us2, HSV-2 Us2, or mono- and polyubiquitin (FK2). (C) HSV-2 Us2 coimmunoprecipitates with ubiquitin-conjugated proteins from infected cells. Vero cells were infected with WT HSV-2 or a ΔUs2 virus for 6 h. Cells were collected, and lysates were incubated overnight with a monoclonal antibody against mono- and polyubiquitin (FK2). Proteins were pulled down using protein G agarose and were subjected to Western blotting for detection of the presence of Us2, ICP5, ICP27, UL21, and gD. Asterisks indicate the Ig heavy chain. (D) HSV-2 Us2 interacts directly with ubiquitin. Vero cells were infected with WT HSV-2 for 6 h. Cell lysates were prepared and were incubated overnight with ubiquitin-conjugated beads. Interacting proteins were subjected to Western blotting and were probed with an antibody against Us2. G, protein G agarose (negative control); mono, monoubiquitin-conjugated agarose; K48, K48-linked tetraubiquitin-conjugated agarose; K63, K63-linked tetraubiquitin-conjugated agarose. (E) HSV-2 Us2 interacts with ubiquitin in the absence of other viral proteins. An empty vector (pFlag-CMV2) or N-terminally Flag-tagged Us2 was transfected into 293T cells. At 24 h posttransfection, cells were collected in lysis buffer, and lysates were incubated overnight with anti-Flag beads at 4°C. Immunoprecipitated proteins were eluted using 3×Flag peptides. Eluates were incubated overnight with either protein G agarose or monoubiquitin-conjugated beads at 4°C. Us2 bound to the beads was detected by Western blotting using anti-Flag antibodies. G, protein G agarose; mono, monoubiquitin-conjugated beads.