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

Our previous studies have shown that TRIM56 is an antiviral host factor against several different RNA viruses, among which include 3 members of the Flaviviridae, DENV2, YFV, and BVDV [24, 25] . To determine whether TRIM56 impacts ZIKV fitness, we conducted infection experiments in HeLa-FitA2-T56 cells with Tet-inducible expression of HA-tagged TRIM56 we developed previously [34] . In these cells robust expression of HA-TRIM56 protein could be turned on upon addition of doxycycline (Dox) to culture medium (Fig 1A, compare lanes 2 vs l). When infected with ZIKV-MR766, cells without HA-TRIM56 expression harbored abundant viral E protein at 72 h.p.i. (Fig 1A, lanes 3 and 5) . In contrast, the level of ZIKV E protein was profoundly reduced in cells with Dox-induced HA-TRIM56 expression (Fig 1A, lanes 4 and 6) . Consistent with the viral protein data, progeny virus titers in culture supernatants were significantly lower in cells induced for HA-TRIM56 expression than those cultured in the absence of Dox (Fig 1B, compare bars 4 vs 3, a 11.7-fold decrease at MOI 0.5). Immunofluorescence staining revealed that the percentage of cells positive for ZIKV E antigen was substantially lower in cells with Dox treatment than those without (Fig 1C) . To ensure this is not a cell-type-specific phenomenon, we assessed the anti-ZIKV activity of TRIM56 in HEK293--FIT-T56 cells that express HA-TRIM56 in a Tet/Dox-inducible manner [24] . As shown in Fig 1D, the results mirrored those obtained in HeLa-FitA2-T56 cells. In addition to the Tet-inducible expression system, a constitutive overexpression strategy was also utilized to examine the impact of TRIM56 on ZIKV infection. Retrovirus-mediated ectopic expression of Flag-HAtagged TRIM56 (FH-T56) curtailed viral E protein expression in HEK293-T3Y cells infected by ZIKV (Fig 1E, compare lanes 4 vs 3, and lanes 6 vs 5). Taken together, these results reveal that ectopic expression of TRIM56 inhibits the propagation of an African strain of ZIKV.