Regulation of host innate immunity by HSV ICP-0

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HSV infection activates a robust innate response through engagement of multiple pattern recognition receptors (PRR) including TLR2, TLR9. Signaling events downstream of these receptors, through activation of NF-kB and IRF3 responsive genes, initiate an innate inflammatory response which inhibits viral replication and spread. Multiple lines of evidence suggest that inhibition of host innate immunity is key to HSV survival, replication and ability to establish life-long latency. By differentially comparing the innate response to two HSV viral vectors, helper-free HSV amplicon that does not encode any viral proteins and helper-containing HSV amplicon that expresses four HSV immediate early proteins, ICP-0, ICP-22, ICP-27 and ICP-47, in primary human tumor cells, and in TLR2 and TLR9 expressing HEK293 cell lines, we identified HSV immediate early protein ICP0 as a negative regulator of TLR signaling. In transfection experiments, ICP-0 inhibits signaling downstream of both MyD-88 dependent (TLR2, 4, 7, 8, 9) as well as MyD-88 independent (TLR3) TLR receptors. ICP0 exerts this activity by targeting TRAF6 for de-ubiquitination, an activity dependent on ICP0 association with the de-ubiquitinating enzyme USP7 (HAUSP) and independent of its E3 ligase function, which has previously been shown to inhibit IRF3 signaling. ICP-0 expression vectors harboring point mutations/deletions that target the RING domain E3 ligase function or binding of ICP-0 to USP7 showed decreased ability to inhibit either IRF3 or TRAF6 signaling, respectively. Knockdown of endogenous USP7 by RNAi severely impaired ICP0-mediated inhibition of TLR signaling. In contrast, over-expression of USP-7, in the absence of ICP-0 was sufficient to inhibit TRAF6- signaling. These results shed light on a novel aspect of ICP-0 biology, namely its ability to inhibit TLR signaling by recruiting USP-7 to de-ubiquitinate TRAF6. Given the pivotal role of TRAF6 and IRF3 at the cross-roads of various innate signaling pathways, and ICP-0 known ability to inhibit IRF3, these studies identify ICP-0 as a master regulator of innate immunity to HSV infection. Ability of ICP-0 to inhibit both IRF3 and NF-kB signaling pathways, the former through its E3 ligase function and the latter through its association with USP-7, may afford HSV comprehensive protection from host immunity during repeated cycles of lytic infection and reactivation from latency. The work also identifies a rare example of how two seemingly contradictory biologic functions resident within ICP-0, namely E3 ligase activity at the Nh2 terminus and de-ubiquitinating activity through association of ICP-0 COOH terminus with USP-7, may cooperate to silence multiple signaling pathways integral to the innate response to infection. We have also identified a previously unknown function for USP7 in regulating innate signaling beyond its known function as a regulator of p53.