Figure 6.

HIRA trimerization is required for HBV minichromosome establishment. (A) HepG2^hNTCP cells were transfected twice with siHIRA or siCTL and then transfected with plasmids encoding for either WT HIRA (pEYFP-N1-HIRA) or for a trimerization-incompetent HIRA mutant (pEYFP-N1-HIRA W799A D800A) before inoculation with HBV. The cells were harvested for analysis 2 dpi. (B) HIRA messenger RNA (mRNA) and protein expression after siRNA transfection and transcomplementation was determined by real-time qPCR and Western blot. β-actin served as Western blot loading control. HIRA mRNA was normalized over housekeeping GUSb gene expression and expressed as relative to the control treated only with the TRA. (C) cccDNA levels at 2 dpi were measured by qPCR. The cccDNA amount was normalized over β-globin quantity and then expressed as relative to the control treated only with TRA. (D) Schematic representation indicating that HIRA trimerization, required for H3.3 deposition, is necessary for full PF-rcDNA to cccDNA conversion in living infected hepatocytes. Graphs represent the means ± SEM of at least 3 independent experiments. The 2-tailed P value was calculated for a risk threshold of .05 using the 2/K sample permutation test with Monte Carlo resampling approximation. ∗∗P < .01, and ∗∗∗P < .001.