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

The retroviral vectors encoding C-terminally Flag-tagged, wild-type (WT) human TRIM56, the E3 Ub ligase-deficient CC21/24AA mutant, and a deletion mutant lacking C-terminal aa 693 to 750, respectively, in the pCX4bsr backbone, have been described previously [25] . N-terminally Flag-and HA-tandem tagged human TRIM56 (FH-T56) was inserted into the pCX4pur retroviral vector backbone, to yield pCX4pur-FH-T56. To express a recombinant TRIM56 protein fragment in E. coli for polyclonal antibody production and protein-RNA interaction assay, we inserted a cDNA fragment encoding the C-terminal 392 aa of human TRIM56 (T56-C392) into pMAL-c4x (New England Biolabs). The resultant plasmid vector, designated pMAL-c4x-T56-C392, allowed expression and subsequent purification of an MBP-T56-C392 fusion protein in E. coli. The plasmid encoding serotype 1 DENV replicon, pACYC-DENV1-Rluc2A-Rep [30] , was obtained from Ju-Tao Guo and Jinhong Chang (Baruch S. Blumberg Institute) with permission of Pei-Yong Shi (University of Texas Medical Branch). The NS4B coding sequence of ZIKV (MR766 strain) was amplified by PCR from the cDNA of virally infected Vero cells and ligated into pEF6/V5-His-TOPO (Invitrogen) to yield the pEF6-ZIKV-NS4B-V5His6 construct, from which NS4B would be expressed as a protein fused to C-terminal V5-His6 epitope tags. All plasmids were verified by Sanger DNA sequencing [31] .