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

HeLa-Flp-In T-REx-ACE2 (referred to as HeLa-FitA2) cells with tetracycline (Tet)-inducible expression of 2xHA-tagged, WT, E3 Ub ligase-deficient CC21/24AA mutant, or C-terminal 693-750 aa deletion mutant of TRIM56 have been described in a previous study [34] . Tetinducible expression of HA-TRIM56-WT had also been established in HEK293-Flp-In T-Rex (referred to as HEK293-FIT, Invitrogen) cells [24] . HEK293-T3Y cells that stably express very low levels of C-terminally YFP-tagged TLR3 were kindly provided by Kate Fitzgerald (University of Massachusetts Medical School). To stably express Flag-and HA-tandem tagged TRIM56 (FH-T56), we transduced HEK293-T3Y cells with a replication-incompetent retrovirus packaged from pCX4pur-FH-TRIM56. Following selection with puromycin, surviving cells were pooled, designated as HEK293-T3Y-FH-T56 cells, and used for subsequent analyses. The blasticidin-resistant, HEK293 cells with stable, constitutive expression of Flag-tagged WT TRIM56 or the E3 ligase-dead CC21/24AA mutant have been described [24] . To create HEK293 cells with constitutive expression of the TRIM56-Flag mutant lacking C-terminal 693-750 residues, cells were transduced with a pCX4bsr-derived retroviral vector carrying the mutant TRIM56 cDNA, followed by stable selection with blasticidin. Similar strategy was adopted to establish SK-N-SH-, HEK293T-and No-Dice (4-25)-derived cell populations with constitutive expression of TRIM56-Flag or the empty pCX4bsr vector, as controls.