Schematic representation of signaling pathways involved in dsRNA/poly(I · C)-induced apoptosis. See the text for details and abbreviations.

The HSV-1 R1-null mutant ICP6Δ is defective in blocking apoptosis induced by poly(I · C). (A and B) HeLa cells were mock infected or infected with HSV-1 KOS (KOS) or HSV-1 ICP6Δ (ICP6Δ) for 8 h before the addition of control medium (ctl), CHX, poly(I · C), CHX+poly(I · C), or lipo+poly(I · C). The cells were collected after 6 h. Cytoplasmic cell lysates were analyzed by immunoblotting for caspase 8 (Casp-8) (MAb 1C12) and β-actin protein content (A) and tested for caspase 3/7 (Casp-3/7) activity (B). The immunoblots and caspase activities (mean plus SEM; n = 6; **, P < 0.01) are representative of 3 independent experiments performed in duplicate. (C) Levels of c-FLIP_L and c-FLIP_S isoforms and c-IAP1/2 decrease after HSV infection. HeLa cells were infected with either HSV-1 KOS (KOS) or HSV-1 ICP6Δ (ICP6Δ). They were collected at the indicated times postinfection (hpi), and total protein extracts were immunoblotted for HSV R1, c-IAP1, c-IAP2, c-FLIP, HSV R2, ICP0, and β-actin protein content determination. The immunoblots are representative of 2 independent experiments performed in duplicate.

HSV-2 R1 inhibits apoptosis induced by TRIF or RIP1 overexpression. HeLa cells were mock infected or infected with AdTR5CuO (Ad ctl) or AdCMV5R1 (Ad HSV-2 R1). After 8 h, the cells were cotransfected with pEGFP C1 mixed with empty vector (EV) or expression plasmids encoding TRIF-FLAG, TRIFΔC-FLAG, or myc-RIP in the presence or absence of Z-VAD-fmk. (A) The percentages of GFP-positive adherent cells with healthy morphology were scored after 24 h. (B) Cytoplasmic cell lysates were immunoblotted for HSV-2 R1, caspase 8 (Casp8) (MAb 1C12), β-actin, and myc-tagged and FLAG-tagged protein content determination. Myc-RIP1_C indicates a processed form of myc-RIP1. The percentages of GFP-positive adherent cells and immunoblots (mean plus SEM; n = 3; ***, P < 0.001) are representative of 3 independent experiments.

HSV-1 and HSV-2 infections protect HeLa cells against poly(I · C)-induced apoptosis. (A) One day after plating, HeLa cells were treated with control medium (ctl), CHX, poly(I · C), CHX+poly(I · C), or lipo+poly(I · C). Phase-contrast microphotographs were taken 6 h later. (B) HeLa cells were mock infected or infected with either HSV-1 strain KOS (HSV-1) or HSV-2 strain HG-52 for 8 h before the addition of control medium (ctl), CHX, poly(I · C), CHX+poly(I · C), or lipo+poly(I · C). The percentages of apoptotic cells were scored after 6 h (mean plus SEM). (C) Cells prepared for panel B were harvested, and the cytoplasmic lysates were tested for caspase 3/7 (Casp-3/7) activity (mean plus SEM). (D) HeLa cells were mock infected or infected with HSV-1 strain KOS (HSV-1) or HSV-2 strain HG-52 (HSV-2) for increasing periods before the addition of CHX, CHX+poly(I · C), or control medium (ctl medium). The percentages of apoptotic cells were scored 6 h after treatment (mean ± SEM).

HSV-1 R1 and HSV-2 R1 inhibit caspase activation induced by poly(I · C). HeLa cells were transfected with pLBPf1-GST (pGST) or pLBPf1-GST-R1 (pGST-HSV-1 R1) (A) and pAdCMV5 (pCMV) or pAdCMV5-R1 (pHSV-2 R1) (B). After 48 h, the cells were left untreated (ctl) or treated with CHX, poly(I · C), CHX+poly(I · C), or lipo+poly(I · C). They were treated with CHX plus TNF-α as a positive control for HSV R1 protection. The percentages of apoptotic cells were scored after 6 h (top). Cytoplasmic lysates were immunoblotted for HSV R1, caspase 8 (Casp-8) (MAb 1C12), and β-actin protein content determination (middle) and tested for caspase 3/7 (Casp-3/7) activity (bottom). The percentages of GFP-positive cells evaluated on parallel dishes cotransfected with pEGFP C1 gave transfection efficiency estimates of >80%. The immunoblots, percentages of apoptotic cells, and caspase activities (mean plus SEM; n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001) are representative of 3 independent experiments.

HSV R1s interact with RIP1. (A) HSV-1 R1 coprecipitates with RIP1. HeLa cells were transfected with pLBPf1-GST (pGST) or pLBPf1-GST-R1 (pGST-HSV-1 R1) for 48 h. GST-tagged proteins were precipitated by GST pulldown. Precipitates (GST pulldown) and whole-cell lysates were immunoblotted for HSV-1 R1, caspase 8 (Casp-8) (MAb 1C15), and RIP1 protein content determination. (B) HSV-2 R1 coimmunoprecipitates with RIP1. HeLa cells were infected with AdTR5CuO (Ad ctl) or AdCMV5R1 (Ad HSV-2 R1). After 8 h, the infected cells were transfected with pcDNA3-6myc-RIP1 (myc-RIP1) for 24 h in the presence of Z-VAD-fmk to maintain cell viability. Myc-tagged proteins were immunoprecipitated with anti-myc antibody. Immunoprecipitates (IP: myc) and whole-cell lysates (lysates) were immunoblotted for HSV-2 R1, FADD, and myc-tagged protein content determination. (C) A large part of the N-terminal, but not the C-terminal, domain of HSV-2 R1 is dispensable for interaction with RIP1 and caspase 8. A549-tTA cells were infected with AdTR5-R1-GFP (Ad R1-GFP), AdTR5-R1(1-834)-GFP (Ad R1(1-834)-GFP), or AdTR5-GFP (Ad GFP) for 24 h. The GFP-tagged proteins were immunoprecipitated with polyclonal anti-GFP antiserum. Immunoprecipitates (IP: GFP) and whole-cell lysates (lysates) were immunoblotted for caspase 8 (MAb 1C15), RIP1, and GFP-tagged protein content determination. (D) HSV-1 R1 and HSV-2 R1 coimmunoprecipitate with RIP1 in the context of HSV infection. HeLa cells were infected with HSV-1 (strain KOS) or HSV-2 (strain HG-52) for 8 h. RIP1 was immunoprecipitated using anti-RIP1 (clone 38) MAb. Immunoprecipitates (IP: RIP1) and whole-cell lysates (lysates) were immunoblotted for HSV R1s, RIP1, and caspase 8 protein content determination. (E) HSV-2 R1 inhibits the interaction between RIP1 and TRIF. HeLa cells were infected with AdTR5CuO (Ad ctl) or AdCMV5R1 (HSV-2 R1) and 8 h later cotransfected with a plasmid encoding myc-RIP1 and a plasmid expressing TRIF-FLAG in the presence of Z-VAD-fmk. The FLAG-tagged proteins were immunoprecipitated with anti-FLAG (M2) MAb. The immunoprecipitate (IP: FLAG) and whole-cell lysate (lysates) protein contents were analyzed by immunoblotting with the polyclonal anti-R1 serum 168R1 and with anti-RIP1 (G322.2) and anti-FLAG (M2) MAbs. As a precipitation control (ctl), precleared lysates were incubated with protein G Sepharose beads without antibody. The immunoblots are representative of at least 2 experiments performed in duplicate.

TRIF silencing inhibits caspase activation induced by extracellular poly(I · C) in mock- and ICP6Δ-infected cells. (A) TRIF siRNA efficiently decreases TRIF expression. HeLa cells were transfected with control siRNA (siCTL) or TRIF siRNA (siTRIF) using Lipofectamine 2000. After 48 h, the cells were mock infected or infected with HSV-1 ICP6Δ for 8 h. Total protein extracts were immunoblotted for HSV R1, HSV R2, ICP0, TRIF, and β-actin protein content determination. (B and C) Effect of TRIF silencing on caspase activation. HeLa cells were transfected with siRNAs and subsequently mock infected (B) or ICP6Δ infected (C) for 8 h before the addition of control medium (ctl), CHX, poly(I · C), or CHX+poly(I · C). The cells were collected after 6 h. Cytoplasmic cell lysates were immunoblotted for caspase 8 (Casp-8) (MAb 1C12) and β-actin protein content determination (top) and tested for caspase 3/7 (Casp-3/7) activity (bottom). The immunoblots and caspase activities (mean plus SEM; n = 6; ***, P < 0.001) are representative of 3 independent experiments performed in duplicate.