1D). A previous study has shown an inhibitory effect of MxA on the nucleocytoplasmic export of HBV mRNA.11 We therefore checked the expression and the cytoplasmic/nuclear distribution of intracellular HBV RNAs in HepG2.2.15 cells. Results from real-time PCR demonstrated that neither the total RNAs nor its intracellular distribution was altered by MxA or each of the two mutants, as measured at 24 hours after transfection http://www.selleckchem.com/products/z-ietd-fmk.html (Supporting Fig. 2A,B). Consistently, results of Western blot showed that the intracellular HBcAg protein level was not remarkably influenced (Supporting Fig. 2C). Taken together, these results suggest that in HepG2.2.15 cells, MxA GTPase activity independently inhibits HBV replication without altering

the cytoplasmic/nuclear HBV mRNA distribution Trametinib solubility dmso and HBcAg level, at least in the early stage of MxA expression. To investigate the mechanism underlying the anti-HBV effect of MxA, we observed the location of HBcAg, the core protein of HBV, in hepatoma cells expressing HBV plasmid and CFP-tagged MxA. Immunofluorescence images revealed that in HBV-transfected Huh7 cells without CFP-MxA expression, HBcAg was spread throughout the cytoplasm and the nucleus in a small punctate pattern (data not shown). Strikingly, in CFP-MxA–overexpressing cells, HBcAg colocalized

with CFP-MxA to generate large granular structures in the cytoplasm (Fig. 2A). To further verify the colocalization of MxA and HBcAg, we overexpressed the two proteins in a nonhepatoma cell type. In living Vero cells, YFP-HBcAg accumulated in the perinuclear region, overlapping with either the wild-type or the mutant CFP-MxA Etoposide cell line (Fig. 2B). Colocalization of MxA with HBcAg prompted us to look for evidence of their possible interaction. First, we determined whether MxA and HBcAg could undergo

coprecipitation. In Huh7 cells expressing Flag-MxA and YFP-HBcAg, immunoprecipitation of HBcAg using GFP antibody resulted in coprecipitation of Flag-MxA (Fig. 2C). The formation of the MxA-HBcAg complex was not dependent on the GTPase activity of MxA, because the Flag-L612K and Flag-K83A mutants of MxA were coimmunoprecipitated with YFP-HBcAg to a similar degree. We then tested whether exogenous HBcAg could interact with endogenous MxA, or exogenous MxA with endogenous HBcAg, because a previous study showed that in HBV-expressing HepG2.2.15 cells, IFN is unable to induce MxA expression.14 By treatment of Huh7 cells expressing Flag-HBcAg with IFNα, or transfection of HepG2.2.15 cells with Flag-MxA, we found that Flag-HBcAg coprecipitated IFNα-induced MxA (Fig. 2D), while Flag-MxA coprecipitated endogenous HBcAg (Fig. 2E), indicating a specific interaction between HBcAg and MxA. Finally, we performed fluorescence resonance energy transfer (FRET) experiments, which detect the proximity of interacting proteins. In living Vero cells expressing CFP-MxA and YFP-HBcAg, the proteins were first confirmed to colocalize to perinuclear structures.