Immunoprecipitation and immunoblot assays.

Confluent Vero cells in T75 flasks were infected with the double-tagged recombinant virus VC1 (gK-V5 and UL20-FLAG), DC474-480, or F strain virus at an MOI of 2. At 24 hpi, the infected cells were lysed with NP-40 cell lysis buffer (Life Technologies) supplemented with protease inhibitor tablets (Roche). The samples were centrifuged at 13,000 rpm for 10 min at 4°C. The supernatants were then used for immunoprecipitation. The proteins from virus-infected cells were immunoprecipitated using protein G magnetic Dynabeads according to the manufacturer's instructions (Invitrogen). Briefly, the beads were bound to their respective antibodies and left on a nutator for 10 min, followed by the addition of cell lysates. The lysate-bead mixture was kept on the nutator for 10 min at room temperature and subsequently washed three times with phosphate-buffered saline (PBS). The protein was eluted from the magnetic beads in 40 μl of elution buffer and used for immunoblot assays. Sample buffer containing 5% β-mercaptoethanol was added to the protein and heated at 55°C for 15 min. Proteins were resolved in a 4-to-20% SDS-PAGE gel and immobilized on nitrocellulose membranes. Immunoblot assays were carried out using monoclonal mouse anti-FLAG antibody (Sigma-Aldrich, Inc., St. Louis, MO), monoclonal mouse anti-V5 antibody (Invitrogen), mouse monoclonal anti-VP5 antibody, horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies (Abcam, Inc., Cambridge, MA), polyclonal rabbit anti-UL37 antibody (a gift from Frank J. Jenkins, University of Pittsburgh Cancer Institute), and HRP-conjugated goat anti-rabbit and anti-mouse antibodies (Abcam, Inc., Cambridge, MA).