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 assess whether TRIM56 directly interacts with ZIKV RNA, we first purified MBP-tagged T56-C392 (comprising the C-terminal 392 aa of human TRIM56) protein from E. coli using the pMAL Protein Fusion and Purification System (New England Biolabs, E8000S, USA). To produce a control protein, we purified MBP along with a short stretch of the downstream polylinker (MBP-polylinker) from E. coli transformed with the empty vector pMAL-c4x. ZIKV RNA was extracted using TRIzol from virions in high-titer ZIKV stocks. Subsequently, two micrograms of MBP-polylinker or MBP-T56-C392 protein were incubated with 0.5 μg of ZIKV RNA at room temperature for 30 min, followed by pull-down of MBP-tagged proteins using amylose resin. After three washes, the resins were divided equally into two fractions, which were subjected to RNA isolation by TRIzol and protein extraction by boiling in SDS sample buffer, respectively. Quantification of ZIKV RNA levels was performed by qPCR as described above. MBP-polylinker and MBP-T56-C392 in the protein samples were probed by immunoblotting using rabbit anti-MBP pAb (New England Biolabs) and rabbit anti-TRIM56 pAb (Bethyl Labs).