Direct activation of RIP3/MLKL-dependent necrosis by herpes simplex virus 1 (HSV-1) protein ICP6 triggers host antiviral defense

Abstract

The receptor-interacting kinase-3 (RIP3) and its downstream substrate mixed lineage kinase domain-like protein (MLKL) have emerged as the key cellular components in programmed necrotic cell death. Receptors for the cytokines of tumor necrosis factor (TNF) family and Toll-like receptors (TLR) 3 and 4 are able to activate RIP3 through receptor-interacting kinase-1 and Toll/IL-1 receptor domaincontaining adapter inducing IFN-β, respectively. This form of cell death has been implicated in the host-defense system. However, the molecular mechanisms that drive the activation of RIP3 by a variety of pathogens, other than the above-mentioned receptors, are largely unknown. Here, we report that human herpes simplex virus 1 (HSV-1) infection triggers RIP3-dependent necrosis. This process requires MLKL but is independent of TNF receptor, TLR3, cylindromatosis, and host RIP homotypic interaction motif-containing protein DNA-dependent activator of IFN regulatory factor. After HSV-1 infection, the viral ribonucleotide reductase large subunit (ICP6) interacts with RIP3. The formation of the ICP6-RIP3 complex requires the RHIM domains of both proteins. An HSV-1 ICP6 deletion mutant failed to cause effective necrosis of HSV-1-infected cells. Furthermore, ectopic expression of ICP6, but not RHIM mutant ICP6, directly activated RIP3/MLKL-mediated necrosis. Mice lacking RIP3 exhibited severely impaired control of HSV-1 replication and pathogenesis. Therefore, this study reveals a previously uncharacterized host antipathogen mechanism.programmed necrosis | HSV-1 | ICP6 | RIP3 | MLKL C ell death triggered by pathogens is a crucial component of mammalian host-defense system. Apoptosis, a predominant programmed cell death in mammals, functions as an effective host-defense mechanism for preventing pathogen replication. Apoptosis is initiated by either mitochondria or cell-death receptors, and it is executed by a group of cysteine proteases called caspases (1). The apoptotic pathway can be subverted by pathogen-encoded apoptotic suppressors such as caspase inhibitors (2). Recent studies have revealed that caspase inhibition can lead to alternative activation of necrosis, releasing the damage-associated molecular patterns (DAMPs) signal to trigger the activation of the host immune system (3, 4).Cytokines of the TNF family are classical inducers of programmed necrosis that are morphologically characterized by the swelling of intracellular organelles and disrupted plasma membranes. Programmed necrosis triggered by death cytokines such as TNF, also known as necroptosis (5-7), is tightly regulated by receptor-interacting kinase-1 (RIP1) (8), its deubiquitin enzyme cylindromatosis (CYLD) (9), and receptor-interacting kinase-3 (RIP3) (10-12). The RIP homotypic interaction motif (RHIM) domains of RIP1 and RIP3 are required for the formation of the RIP1-RIP3 complex that is called a necrosome (13). Recently, mixed lineage kinase domain-like (MLKL) protein has been identified as a functional substrate of RIP3 kinase (14, 15). Upon phosphorylation, ...

Functional Comparison of Molluscum Contagiosum Virus vFLIP MC159 with Murine Cytomegalovirus M36/vICA and M45/vIRA Proteins

“…A possible explanation for this discrepancy is that the association of MC159 with RIP1 is sufficient for inhibition of TNF-␣-induced necroptosis in human but not in mouse cells. Interestingly, a species-specific necroptosis inhibition has been reported for the herpes simplex virus 1 (HSV-1) ICP6 and the HSV-2 ICP10 proteins: ICP6 and ICP10 inhibit necroptosis in human but not in mouse cells (45,46,53). ICP6, ICP10, and M45 are homologous proteins, and all three possess a RHIM (54), which is required for the inhibition of RIP3-dependent necroptosis (29,45,46).…”

Suppression of RIP3-dependent Necroptosis by Human Cytomegalovirus

“…Recently, the RHIM-targeted suppressor strategy has been extended to HSV, where the large subunit of ribonucleotide reductase (R1) suppresses necroptosis in human cells (41) in addition to its role in suppressing Casp8-dependent apoptosis (50). Surprisingly, HSV triggers necroptosis in mouse cells, and this vulnerability is influenced by R1 RHIM signaling (51,52). In contrast, murine CMV vIRA suppresses cell death in both mouse and human cells (41).…”

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

“…M45/vIRA mutant MCMV triggers programmed necrosis by inducing an interaction between RIP3 and the DNA-dependent activator of IFN regulatory factor (DAI) (24). Unlike VV and MCMV, herpes simplex virus 1 (HSV-1) infection naturally activates mouse RIP3 (mRIP3)/mMLKL-dependent necrosis in mouse cells independently of TNFR, TLR3, and DAI (25,26). During HSV-1 infection, RIP3 is activated by the assembly of a complex with the RHIM-containing viral protein ICP6, the large subunit (R1) of ribonucleotide reductase (RR), leading to MLKL activation and necrosis of host cells (25,26).…”

“…Unlike VV and MCMV, herpes simplex virus 1 (HSV-1) infection naturally activates mouse RIP3 (mRIP3)/mMLKL-dependent necrosis in mouse cells independently of TNFR, TLR3, and DAI (25,26). During HSV-1 infection, RIP3 is activated by the assembly of a complex with the RHIM-containing viral protein ICP6, the large subunit (R1) of ribonucleotide reductase (RR), leading to MLKL activation and necrosis of host cells (25,26). RIP3-deficient mice showed severely impaired control of HSV-1 replication and pathogenesis (25).…”

“…During HSV-1 infection, RIP3 is activated by the assembly of a complex with the RHIM-containing viral protein ICP6, the large subunit (R1) of ribonucleotide reductase (RR), leading to MLKL activation and necrosis of host cells (25,26). RIP3-deficient mice showed severely impaired control of HSV-1 replication and pathogenesis (25). Although HSV-1 is a common human herpesvirus, it remains unclear precisely how HSV-1 modulates programmed necrosis in human cells.…”