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. 2019 Apr 18;47(12):6411–6424. doi: 10.1093/nar/gkz278

Figure 2.

Figure 2.

TNRC6 proteins are required and functionally redundant for optimal HCV replication. (A) HJ3-5/GLuc2A reporter virus genome, showing GLuc2A insertion between p7 and NS2A coding regions. (B) HCV reporter virus replication measured by GLuc secretion in Huh-7.5 cells transfected with siRNAs depleting single TNRC6 paralogs (see Supplementary Figure S3A). Results shown are means of n = 3 technical replicates ± s.d., and are representative of two independent experiments. *TNRC6C versus Ctrl siRNAs P ≤ 0.029 at 72 h by two-way ANOVA with Sidak’s multiple comparison test. (C) Reporter virus replication in cells depleted of two or more TNRC6 proteins (see Supplementary Figure 3A). Results shown are means of three technical replicates ± s.d. and are representative of two independent experiments. ****TNRC6 versus Ctrl siRNAs, P ≤ 0.0001; ++++TNRC6B/C versus TNRC6A/B or TNRC6A/C siRNAs, at 48 and 72 h by two-way ANOVA with Sidak’s multiple comparison test. (D) Immunoblots of TNRC6 proteins 48 h after siRNA transfection. Ago2 protein was blotted for comparison. β-Actin was a loading control. (E) Cell-associated HCV RNA (normalized to β-actin mRNAabundance) 72 h after infection of TNRC6-depleted Huh-7.5 cells with cell-free HJ3-5 virus at a multiplicity of 0.1. HCV RNA abundance in siCtrl cells was arbitrarily set to 100. n = 3 from two independent experiments, one with two technical replicates (shaded symbols). *P < 0.05 versus Ctrl siRNA (adjusted P = 0.0178 for TNRC6B/C, and 0.0384 for TNRCA/B/C) by one-way ANOVA with Dunnett’s correction for multiple comparisons. (F) Left panel: Domain structure of TNRC6B with the amino-terminal fragment (5′F), carboxy-terminal fragment (3′F), T6B polypeptide segment indicated. The site targeted by siTNRC6B is also shown. Right panel: Immunoblot showing the expression of control eYFP and indicated eYFP-T6B polypeptides. (G) Reporter virus replication in Huh-7.5 cells overexpressing the indicated proteins. Results shown are means of three biological replicates ± s.d. ****P<0.0001 by two-way ANOVA with Sidak’s multiple comparison test. (H) Left panel: Immunoblots showing the levels of endogenous TNRC6A/B/C and exogenously expressed TNRC6B-5′F and 3′F. TNRC6B-5′F is not recognized by TNRC6 antibody but can be detected by Flag antibody. Right panel: Reporter virus replication in cells transfected with control siRNA or TNRC6B/C siRNAs and overexpress indicated proteins. Results shown are means of three experiments ± s.d. ****P<0.0001 by two-way ANOVA with Sidak’s multiple comparison test.