Journal: Journal of Virology

Article Title: Herpes Simplex Virus 1 Tegument Protein US11 Downmodulates the RLR Signaling Pathway via Direct Interaction with RIG-I and MDA-5

doi: 10.1128/JVI.06713-11

Figure Lengend Snippet: US11 interacts with both RIG-I and MDA-5 in transfected and HSV-1-infected cells. (A, B, C, and D) US11 associates with both RIG-I and MDA-5 in transfected cells. HEK293T cells (∼5 × 106) were cotransfected with 10 μg of plasmid US11-HA and with 10 μg of plasmid pEF-Flag-RIG-I, encoding full-length RIG-I (A and C), or with plasmid pEF-Flag-MDA-5, encoding full-length MDA-5 (B and D), respectively. At 36 h after transfection, cells were lysed and clarified supernatants were left untreated (A and B) or treated with RNase A at 150 μg/ml (C and D). The samples were then subjected to immunoprecipitation assays using anti-HA MAb (IP: HA) or nonspecific mouse monoclonal antibody (IgG2b). Cell lysates and immunoprecipitated proteins were separated in denaturing 12% polyacrylamide gels and transferred to nitrocellulose membranes. The transferred proteins were probed with anti-HA and anti-Flag MAbs. (E and F) US11 interacts with overexpressed RIG-I and MDA-5 in HSV-1-infected cells. HEK293T cells were transfected with pEF-Flag-RIG-I (E) or pEF-Flag-MDA-5 (F), respectively. At 20 h after transfection, cells were infected with HSV-1 strain F at an MOI of 10 for 16 h. The cells were then lysed, and the extracts were subjected to immunoprecipitation using anti-Flag MAb (IP: Flag) or nonspecific mouse monoclonal antibody (IgG2b). Precipitates were analyzed by Western blotting using anti-Flag MAb or rabbit anti-US11 pAb. (G and H) US11 interacts with endogenous RIG-I and MDA-5 in HSV-1-infected cells. HEK293T cells were infected with WT or US11 mutant HSV-1 at an MOI of 10 for 16 h. The cells were then lysed, and the extracts were subjected to immunoprecipitation using anti-RIG-I pAb (IP: RIG-I), anti-MDA-5 pAb (IP: MDA-5), or control IgG. Precipitates were analyzed by Western blotting.

Article Snippet: Rabbit anti-RIG-I pAb and rabbit anti-MDA-5 pAb were purchased from GeneTex (San Antonio, TX).

Techniques: Transfection, Infection, Plasmid Preparation, Immunoprecipitation, Western Blot, Mutagenesis

Journal: Journal of Virology

Article Title: Herpes Simplex Virus 1 Tegument Protein US11 Downmodulates the RLR Signaling Pathway via Direct Interaction with RIG-I and MDA-5

doi: 10.1128/JVI.06713-11

Figure Lengend Snippet: US11 blocks the formation of complex between RIG-I and MAVS or between MDA-5 and MAVS. (A and B) HEK293T cells (∼5 × 106) were cotransfected with 10 μg of plasmid pEF-Flag-RIG-I (A) or pEF-Flag-MDA-5 (B) or 10 μg of plasmid pMyc-MAVS and 10 μg of plasmid US11-HA or transfected with empty vector. At 36 h posttransfection, cells were lysed and the clarified supernatants were subjected to immunoprecipitation assays using anti-Myc MAb (IP: Myc) or nonspecific mouse monoclonal antibody (IgG1). US11, MAVS, or RIG-I or MDA-5 was detected by Western blotting using anti-HA, anti-Myc, or anti-Flag MAbs, respectively. (C and D) HEK293T cells were cotransfected with plasmids pMyc-MAVS and pEF-Flag-RIG-I (C) or pEF-Flag-MDA-5 (D), respectively. Twenty hours after transfection, cells were infected with either WT or US11 mutant HSV-1 at an MOI of 10 for another 16 h. The cells were subsequently lysed and subjected to immunoprecipitation assays as described for panel A. Additionally, rabbit anti-US11 pAb was used for detection of US11 expression after either WT or US11 mutant HSV-1 infection.

Article Snippet: Rabbit anti-RIG-I pAb and rabbit anti-MDA-5 pAb were purchased from GeneTex (San Antonio, TX).

Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Infection, Mutagenesis, Expressing