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

TRIM56 targets certain RNA viruses for inhibition at different steps of viral life cycle. It inhibits influenza viruses, YFV and DENV2 by impeding viral RNA synthesis [24, 26] but curbs TRIM56 is an RNA-binding protein that restricts Zika virus human coronavirus (HCoV)-OC43 by acting at the stage of viral packaging/release [24] . To pinpoint where TRIM56 exerts its antiviral action during ZIKV life cycle, we compared the kinetics of intracellular viral RNA accumulation at different times following infection between control and TRIM56-overexpressing cells. At 2 h.p.i., a time point immediately after viral entry and prior to initiation of viral RNA replication, intracellular viral RNA levels were comparable between control HEK293-T3Y cells and cells expressing FH-T56, suggesting that TRIM56 does not affect cellular entry of ZIKV (Fig 5A) . Intracellular viral RNA abundance began to climb at 24 h.p.i. in both cells but did so much more quickly and robustly in cells without FH-T56 expression. For all three time points examined at/after 24 h.p.i., ZIKV RNA replicated to significantly lower levels in FH-T56 cells than in control HEK293-T3Y cells ( Fig 5A) . Confirming the effect was not virus strain-specific, we obtained similar results from experiments using ZIKV-PRVABC59 in lieu of ZIKV-MR766 (Fig 5B) . Importantly, the inhibitory effect of TRIM56 on viral RNA replication was lost upon mutations that abolish the E3 ligase activity or delete its C-terminal portion (Fig 5C) . Furthermore, ZIKV RNAs replicated to significantly higher levels in HeLa-shT56 cells with stable TRIM56 knockdown than in HeLa-shCtrl cells (Fig 5D) , suggesting that endogenous TRIM56 shares the effect with overexpressed TRIM56. We conclude from these data that TRIM56 restricts ZIKV infection by inhibiting viral RNA replication, and that such capacity depends on both its E3 ligase activity and TRIM56 is an RNA-binding protein that restricts Zika virus the integrity of C-terminal portion. Of note, these observations are reminiscent of the effects of TRIM56 on BVDV, YFV, and DENV2 [24, 25] , suggesting a shared antiviral mechanism against flaviviruses. To determine whether this can be said with another flavivirus, we electroporated a luciferase-encoding DENV1 replicon into HEK293-FIT-T56 cells that were repressed (-Dox) or induced (+Dox) for HA-TRIM56 expression and followed up the viral RNA replication kinetics (S6 Fig). Similar to the effects of TRIM56 on BVDV and DENV2 replicons [24, 25] , the result showed that while translation of the input DENV1 replicon RNA (at 3 h post electroporation) was not affected by TRIM56, viral RNA replication at later time points was always significantly lower in cells with HA-TRIM56 induction than those not induced.