2F). Quantification of the fluorescence of CFP-MxA in this region demonstrated a more than 1.22-fold Ridaforolimus molecular weight increase after YFP-HBcAg photobleaching,

whereas no increase was found in the two control groups YFP/CFP-MxA and YFP-HBcAg/CFP (data not shown), indicating an evident energy transfer between the two proteins in the perinuclear compartment. Taken together, our data suggest that MxA interacts with HBcAg in living animal cells. To further dissect the biochemical properties of MxA-HBcAg interaction and determine the relevance of the interaction to the anti-HBV activity of MxA, we created different truncated mutants of MxA (Fig. 3A) and tested their association with HBcAg. Huh7 cells were transfected with Flag-HBcAg and Myc-tagged full-length MxA

or each of the truncated mutants, and the associations were checked by coimmunoprecipitation. We found that MxA deletion mutants either lacking the N-terminal GTP-binding domain, which contains the self-assembly sequence (MxAΔN, 359-662aa), or lacking the C-terminal leucine zipper region (MxAΔC, 1-574 aa), retained the ability to interact with HBcAg as demonstrated by coprecipitation with Flag-HBcAg (Fig. 3B). Interestingly, a MxA deletion mutant lacking the central interactive region (MxAΔCID) was not precipitated by Flag-HBcAg, indicating an essential role of this domain in mediating the MxA-HBcAg association (Fig. 3B). We also coexpressed YFP-HBcAg and CFP-tagged each of the truncated mutants ITF2357 molecular weight to observe the formation of the protein aggregates. We found that, well-correlated with the results of immunoprecipitation, CFP-MxAΔC and CFP-MxAΔN, but not the CFP-MxAΔCID, colocalized with YFP-HBcAg to form large perinuclear aggregates (Fig. 3C), indicating morphologically a requirement for the CID

domain in the generation of MxA-HBcAg complexes. Finally, 上海皓元医药股份有限公司 we assessed the effects of the truncated MxA mutants on HBV replication by measuring the encapsulated viral DNA in the culture medium of HepG2.2.15 cells. Clearly, overexpression of either Myc-MxAΔC or Myc-MxAΔN dramatically decreased HBV DNA level, mimicking that of wild-type Myc-MxA. In contrast, no evident suppression was detected in cells expressing Myc-MxAΔCID (Fig. 3D). Therefore, our results suggest that the CID domain of MxA is the responsive region in mediating the interaction with HBcAg, and MxA-HBcAg interaction is essential to the anti-HBV function of MxA. Given that MxA interacts with HBcAg to form a complex in the perinuclear compartment, and this interaction is required for the anti-HBV activity of MxA, we then aimed at investigating the effect of MxA on the intracellular kinetics of HBcAg. To address this, we performed fluorescence recovery after photobleaching (FRAP) in living cells.