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

ZIKV-infected HEK293 cells expressing control vector, or Flag-tagged, WT or mutant TRIM56, respectively, were washed twice with ice-cold PBS and lysed in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, and protease inhibitor cocktail (Sigma). The cell lysates were incubated at 4˚C for 30 min, clarified by centrifugation at 12, 000 g at 4˚C for 15 min, and their protein concentrations were adjusted to 1 μg/μl. For RNP-IP, 400 μg of cell lysates were incubated with the anti-Flag mAb at 1:500 at 4˚C overnight. Subsequently, RNA-protein-antibody complexes were captured by incubation with protein A/G agarose beads (Santa Cruz Biotech). After three washes, the beads were divided equally into two fractions, which were subjected to RNA isolation by TRIzol and protein extraction by boiling in SDS-sample buffer, respectively. The qPCR analysis of ZIKV RNA levels and immunoblotting of TRIM56 were performed as described above.