The E3 ligase TRIM56 is a host restriction factor of Zika virus and depends on its RNA-binding activity but not miRNA regulation, for antiviral function

Yang, Darong; Li, Nan L.; Wei, Dahai; Liu, Baoming; Guo, Fang; Elbahesh, Husni; Zhang, Yunzhi; Zhou, Zhi; Chen, Guo-Yun; Li, Kui

In line with this model, TRIM71 associates with miRNA and Argonaute2 via its NHL domain, thereby regulating miRNA function and gene expression [60] . The NHL domain also targets TRIM71 to cellular mRNAs, leading to repression of gene expression [28] . Additionally, NHL repeats confer binding affinity of other TRIM-NHL proteins to cellular miRNAs and mRNAs [27] [28] [29] . Altogether, these prompted us to determine if miRNA regulation and/or RNA-binding play a part in the anti-ZIKV action of TRIM56. To assess the potential connection of TRIM56 with miRNA, we investigated the impact of TRIM56 on ZIKV infection in Dicer knockout cells that lack the biogenesis of mature miRNAs. We did not observe any influence of host miRNA deficiency on TRIM56-imposed suppression of ZIKV RNA replication, viral protein expression, or progeny virus production, suggesting a miRNA-independent antiviral mechanism. We subsequently tested if TRIM56 could bind to viral RNA in infected cells. Indeed, a specific association between TRIM56 and ZIKV RNA was detected in RNP-IP analysis. In contrast, a TRIM56 mutant lacking aa 693-750, the very C-terminal end portion of the NHL-like domain, was no longer able to capture viral RNA, concomitant with a loss in antiviral activity. Importantly, we have demonstrated that a recombinant TRIM56 fragment composed of the C-terminal 392 aa was able to efficiently capture ZIKV RNA in cell-free reactions, suggesting the TRIM56-viral RNA interaction is direct and does not depend on cellular factors, although we cannot exclude the possibility that certain cellular proteins may fine-tune this interaction in infected cells. Interestingly, the E3 ligase activity, another key anti-ZIKV determinant, was found to be dispensable for TRIM56-ZIKV RNA association. Taken together, these observations imply that the full anti-ZIKV action of TRIM56 necessitates not only the C-terminal RNA-binding region to recognize ZIKV RNA, but also the activity of E3 ligase that catalyzes post-translational modification(s) of pro-viral host (or viral) factors to impede their involvement/function in viral RNA replication (S9 Fig). Given previous reports that TRIM5, TRIM25, and TRIM6, can synthesize unanchored polyubiquitin chains via their E3 ligase activities [61] [62] [63] and that unanchored polyubiquitin chains play a role in the life cycle of an RNA virus (influenza A virus) [64] , there is a possibility that TRIM56 may catalyze the synthesis of unanchored polyubiquitin chains to regulate the flaviviral RNA replication process. We also cannot rule out the possibility that TRIM56 promotes ubiquitin-like modifications, such as sumoylation and ISGylation that were reported with TRIM28 and TRIM25 [65, 66] , respectively, to execute its antiviral function.