USP7 is a 1102 animo acid de-ubiquitinating enzyme (DUB) that belongs to the ubiquitin-specific protease (USP) family. By virtue of its interaction with the tumor suppressor p53 and its negative regulator MDM2, USP7 plays a key role in regulation of cell survival and stress response. USP7 also interacts with two viral proteins, namely HSV-1 ICP0 and EBV EBNA-1, however the precise significance of this has not been fully understood. We have previously shown that HSV-1 ICP0 specifically inhibits TLR-mediated innate response to viral infection. In this work, we further investigated this observation, showing that ICP0 recruits USP7, a nuclear protein to the peri-nuclear zone where the latter interacts with TRAF6 and IKKγ. USP7 interaction with TRAF6 and IKKγ were mediated by its amino terminal TRAF domain and resulted in de-ubiquitination of both TRAF6 and IKKγ. Over-expression of USP7 but not the catalytically-inactive mutant C223S (where the active site cysteine was mutated to serine) inhibited TLR response by de-ubiquitinating both TRAF6 and IKKγ, and this effect was augmented by co-expression of ICP0. On the other hand, RNAi-mediated knock-down of endogenous USP7 augmented TLR response, comparable to that seen with A20 and Cyld knockdown and suggesting a significant degree of redundancy at the level of DUB-mediated regulation of TLR signaling. Confocal microscopy showed that association between ICP0 and USP7 alters the sub-cellular localization of USP7 from nuclear to cytoplasmic where it predominantly accumulates in the peri-nuclear region. We hypothesized that translocation of USP7 from nuclear to cytosolic would bring it into close proximity of its two substrates, TRAF6 and IKKγ. In support of this, over-expression of a modified USP7 encoding a nuclear-export signal (NES) and thus expressed in both cytosol and nucleus was associated with enhanced ability to suppress TLR response and de-ubiquitinate IKKγ. In summary, our results identify a novel function for the de-ubiquitinating enzyme USP7 as well as reveal an additional mechanism by which HSV evades host innate immunity.

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