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

Since the NHL repeat motif of TRIM71 has been shown to bind to mRNA [28] , we investigated whether TRIM56 interacts with ZIKV RNA to hamper viral RNA replication. HEK293 cells expressing control vector (Bsr), WT TRIM56, the E3-ligase-dead AA mutant, or Mut N (deltaaa 693-750) lacking a portion of the C-terminal NHL-like repeat sequence were infected by ZIKV, followed by RNP-IP of TRIM56-RNA complexes and qPCR quantifying the viral RNAs associated with TRIM56. The ectopically expressed, Flag-tagged WT and mutant TRIM56 proteins were all efficiently immunoprecipitated by the anti-Flag antibody (Fig 7A, lanes 2-4) , and no background was detected in negative control (Bsr) cells (lane 1). Immunoblotting detection of ZIKV NS5 protein in input cell lysates confirmed the successful infection of ZIKV (lanes 6-9) and the antiviral effect imposed by WT TRIM56 (lane 7). Immunoblotting of the IP-enriched protein complexes showed no association of TRIM56 with viral NS5 protein or cellular actin (Fig 7A, lanes 1-4) , demonstrating the specificity of the RNP-IP assay. Next, we quantified the ZIKV RNA levels co-immunoprecipitated with TRIM56 or the two TRIM56 mutants. Strikingly, significant enrichment of ZIKV RNAs was observed with WT TRIM56 or the AA mutant, but not Mut N lacking C-terminal aa 693-750 (Fig 7B, compare bars 2 and 4 vs 1, increases of~6-8-fold) . These data suggest that TRIM56 binds to viral RNA in ZIKVinfected cells via its C-terminal portion. Because the AA mutant was as efficient as WT TRIM56 in co-precipitating ZIKV RNAs (Fig 7B) , we conclude that the E3 ligase activity, although indispensable for the antiviral function against ZIKV, is not required for the ability of TRIM56 to associate with viral RNAs.