Herpes Simplex Virus 1 (HSV-1) and HSV-2 Mediate Species-Specific Modulations of Programmed Necrosis through the Viral Ribonucleotide Reductase Large Subunit R1

Abstract

Receptor-interacting protein kinase 3 (RIP3) and its substrate mixed-lineage kinase domain-like protein (MLKL) are core regulators of programmed necrosis. The elimination of pathogen-infected cells by programmed necrosis acts as an important host defense mechanism. Here, we report that human herpes simplex virus 1 (HSV-1) and HSV-2 had opposite impacts on programmed necrosis in human cells versus their impacts in mouse cells. Similar to HSV-1, HSV-2 infection triggered programmed necrosis in mouse cells. However, neither HSV-1 nor HSV-2 infection was able to induce programmed necrosis in human cells. Moreover, HSV-1 or HSV-2 infection in human cells blocked tumor necrosis factor (TNF)-induced necrosis by preventing the induction of an RIP1/RIP3 necrosome. The HSV ribonucleotide reductase large subunit R1 was sufficient to suppress TNF-induced necrosis, and its RIP homotypic interaction motif (RHIM) domain was required to disrupt the RIP1/RIP3 complex in human cells. Therefore, this study provides evidence that HSV has likely evolved strategies to evade the host defense mechanism of programmed necrosis in human cells.

Importance: This study demonstrated that infection with HSV-1 and HSV-2 blocked TNF-induced necrosis in human cells while these viruses directly activated programmed necrosis in mouse cells. Expression of HSV R1 suppressed TNF-induced necrosis of human cells. The RHIM domain of R1 was essential for its association with human RIP3 and RIP1, leading to disruption of the RIP1/RIP3 complex. This study provides new insights into the species-specific modulation of programmed necrosis by HSV.

FIG 1

HSV-2 as well as HSV-1 infection directly activates RIP3/MLKL-dependent necrosis in mouse cells. (A) Wild type (WT) and RIP3 knockout (KO) MEFs were infected with a control or HSV-2 at a multiplicity of infection (MOI) of 10 for about 20 h. Identical MOIs were used in MEFs in later experiments. Cell viability analysis was performed as described in Materials and Methods. (B) WT or RIP3 knockout MEF lysates were prepared and subjected to Western blot analysis. (C) L929 cells were transfected with a negative control (NC), mRIP3, or mMLKL siRNA oligonucleotide for 48 h. Then cells were treated as indicated for 15 h, and cell viability was determined. The HSV-1 MOI was 5. T, TNF-α (10 ng/ml); Z, Z-VAD (10 μM). (D) L929 cells were transfected with the indicated siRNA oligonucleotides for 48 h. Then cell lysates were prepared and subjected to Western blot analysis. (E) MEFs stably expressing Flag- and HA-tagged mMLKL were infected with HSV-1 or HSV-2 for 6 h. Cell lysates were prepared for immunoprecipitation with anti-Flag agarose beads. The Flag-mMLKL immunocomplex was then determined by Western blotting of the indicated proteins. IP, immunoprecipitation.

FIG 2

HSV-1 or HSV-2 infection subverts necroptosis in human cells. (A) HT-29 or HT-29-RIP3-shRNA cells were infected with HSV-1 or HSV-2 or treated with TNF-α/Smac mimetic/Z-VAD (40 ng/ml TNF-α, 100 nM Smac mimetic, and 20 μM Z-VAD) for 16 h, and then cell viability was determined. *, P < 0.05 versus the control. (B) HT-29 or HT-29-RIP3-shRNA cells were treated with the indicated virus (MOI of 2.5). Cell lysates were collected at 6 h postinfection and then subjected to Western blot analysis. (C) HT-29 cells were infected with HSV-1 and HSV-2 at the indicated MOI. At 2 h postinfection, cells were treated with dimethyl sulfoxide (DMSO) or TNF-α/Smac mimetic/Z-VAD for 16 h, and then cell viability was determined. *, P < 0.05; **, P < 0.001 versus the control. (D) HT-29 or HT-29-RIP3-shRNA cells were infected with HSV-1 at an MOI of 2.5. At 2 h postinfection, cells were treated with dimethyl sulfoxide or TNF-α/Smac mimetic/Z-VAD for 48 h, and then cell viability was determined. (E) HeLa-hRIP3 cells were infected with the indicated virus. At 2 h postinfection, cells were treated with dimethyl sulfoxide or TNF-α/Smac mimetic/Z-VAD for 16 h, and then cell viability was determined. HeLa-hRIP3 cell lysates were collected after treatment with HSV-1 or HSV-2 (MOI of 2.5). The expression of viral protein VP16 was detected by Western blotting using an anti-VP16 antibody. *, P < 0.05; **, P < 0.001 versus control (F) HT-29 cells were treated with the indicated virus (MOI of 2.5). At 2 h postinfection, cells were treated with dimethyl sulfoxide or TRAIL/Smac mimetic/Z-VAD for 16 h, and then cell viability was determined.

FIG 3

HSV R1 is required to disrupt necrosome formation during necroptosis in human cells. (A) HT-29 cells were infected with the indicated virus (MOI of 2.5). At 2 h postinfection, cells were treated with dimethyl sulfoxide (DMSO) or TNF-α/Smac mimetic/Z-VAD for 16 h, and then cell viability was determined. *, P < 0.05; **, P < 0.001 versus the control. (B) HT-29 cells were infected with the indicated virus (MOI of 2.5). Cell lysates were collected at 6 h postinfection and then subjected to Western blot analysis. (C) HT-29 cells were infected as indicated (MOI of 2.5). At 2 h postinfection, cells were treated with TNF-α/Smac mimetic/Z-VAD for an additional 6 h. Then cell lysates were collected and subjected to Western blot analysis. (D) HeLa-hRIP3 cells were infected as indicated (MOI of 2.5). At 2 h postinfection, cells were treated with TNF-α/Smac mimetic/Z-VAD for an additional 6 h. Cell lysates were collected and used for anti-Flag immunoprecipitation. The Flag-RIP3 immunocomplex was analyzed by Western blotting with the indicated antibody.

FIG 4

Ectopic expression of HSV R1 is sufficient to block TNF-induced necrosis of human cells depending on both RHIM and RR domains. (A) HeLa-hRIP3 cells were transfected with empty vector or an ICP6 or ICP10 DNA plasmid for 24 h. Cells were treated with TNF-α/Smac mimetic/Z-VAD for an additional 36 h, and then cell viability was measured. Data represent the averages ± standard errors for three independent experiments. *, P < 0.05, versus results for the vector. Cell lysates were collected at 24 h posttransfection and subjected to Western blot analysis. (B) Domain structure of ICP6. Full-length ICP6 (amino acids 1 to 1137) contains the N-terminal RHIM domain and the C-terminal RR domain. ICP6(1–101) and ICP6(1–452) contain the N-terminal 101 residues and 452 residues, respectively. ICP6(Δ1–101) lacks residues 1 to 101. The residues 73 to 76 of ICP6 were mutated to four alanine residues. (C and D) HeLa-hRIP3 cells were transfected with the indicated plasmids for 24 h. Cells were treated with TNF-α/Smac mimetic/Z-VAD for an additional 36 h, and then cell viability was measured (C). Data represent the averages ± standard errors for three independent experiments. *, P < 0.05, versus results for the vector. Cell lysates were collected at 24 h posttransfection and subjected to Western blot analysis (D). (E) 293T cells were transfected with the indicated plasmids for 48 h. Cell lysates were collected and immunoprecipitated with anti-Flag agarose beads. The immunoprecipitate was analyzed by Western blotting (WB).

FIG 5

HSV R1 prevents the recruitment of hRIP1 to hRIP3. (A) 293T cells were cotransfected with the Myc-tagged hRIP3 plasmid and a plasmid expressing Flag-tagged ICP10 and the RHIM mutant form of ICP10 (RHIM mut). The residues from 64 to 67 of ICP10 were mutated to four alanine residues. At 48 h posttransfection, cell lysates were collected and used for anti-Flag immunoprecipitation. The Flag-hRIP3 immunocomplex was analyzed by Western blotting with the indicated antibodies. (B) 293T cells were cotransfected with the Flag-tagged hRIP1 plasmid and a plasmid expressing Myc-tagged ICP6, the RHIM mutant, or ICP10. At 48 h posttransfection, cell lysates were collected and used for anti-Flag immunoprecipitation. The Flag-hRIP1 immunocomplex was analyzed by Western blotting with the indicated antibodies. (C) 293T cells were cotransfected with plasmids as indicated. At 48 h posttransfection, cell lysates were collected for anti-Flag immunoprecipitation. The immunocomplex was subjected to Western blot analysis.