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

To determine whether TRIM56 can directly interact with ZIKV RNA, we set up additional binding experiments in cell-free reactions using recombinant TRIM56 protein and viral RNA purified from virions. We expressed and purified from E. coli a TRIM56 fragment comprising the C-terminal 392 aa fused to MBP (MBP-T56-C392), and as a negative control, an MBPpolylinker protein. Their quality and purity were verified by Coomassie blue staining (Fig 7C, left panel) and immunoblotting (right panel) following SDS-PAGE. The recombinant proteins were incubated separately with ZIKV RNA, and thereafter the protein-RNA complexes were recovered by the MBP-tag-binding amylose resin, whereby the associated protein and RNA were isolated. Immunoblotting analysis indicated successful pull-down of the bait proteins, MBP-polylinker and MBP-T56-C392, with comparable efficiency (Fig 7D) . qPCR analysis revealed that, compared with amylose resin alone or the MBP-polylinker control protein pull down groups, there was significant enrichment of ZIKV RNA by recombinant MBP-T56-C392 protein, regardless of viral RNA origin (be it MR766 or PRVABC59) (Fig 7E) . These data demonstrate that TRIM56 can directly associate with ZIKV RNA via its C-terminal portion. This result is also in line with our earlier data obtained from ZIKV-infected cells ( Fig 7A and 7B ).