Preliminary Study On Mechanisms Of Host Protein Alpha-actinin Involving In Hcv Rna Replication

Infection of hepatitis C virus (HCV) continues to be a significant health problem. The chronic hepatitis C patients have a high risk of developing liver cirrhosisi and hepatocellular carcinoma with high mortality rate. Our lab applied yeast two-hybrid system to look for celluar proteins interacting with HCV RNA polymerase (NS5B). Both in vitro and in vivo coimmunoprecipitation and cofocal laser scanning microscopy experiments indicate that NS5B interacted and colocalized with alpha-actinin. RNA interference results show that HCV RNA levels in subgenomic replicon cells significantly decreased after the expression of endogenous alpha-actinin was inhibited. However, the role of host protein alpha-actinin playing in HCV RNA replication has not been elucidated yet.To study the mechanisms of invovlement of alpha-actinin in HCV replication, we first confirmed that alpha-actinin was essential for HCV RNA replication in subgenomic replicon cells and in JFH1-infected cells. By transfecting small interfering RNA against alpha-actinin, we found that the amounts of both HCV RNA and proteins (NS5A and NS3) were reduced in subgenomic replicon cells. After knocking down of alpha-actinin in JFH 1-infected cells, HCV RNA replication and virus production were also significantly reduced. These results suggested that alpha-actinin was indispensable for HCV replication.As alpha-actinin can be regulated by phosphorylation. we investigated if the quantity of phosphorylated alpha-actinin was different between replicon and Huh7 cells and whether phosphorylation level was associated with HCV replication. Co-IP results showed that the phosphorylation level of alpha-actinin in subgenomic replicon cells was lower than that in Huh7 cells. Over-expression of dephosporylation mutant alpha-actinin/Y12F enhanced the expression of NS5A and NS3 in subgenomic replicon cells. Furthermore, in JFH1-infected cells, the amounts of both NS5A and HCV RNA level in cells and supernatant was remarkably increased in the presence of full-length or alpha-actinin/Y12F. These results supported that both alpha-actinin and dephosporylation mutant contributed to HCV RNA replication.Previous experiments showed that HCV replication complexes assemble on detergent resistant membrane domain (DRM) through interactions between viral nonstructural proteins and host proteins. To determine the association of alpha-actinin with DRM, alpha-actinin and mutants were transiently expressed in subgenomic replicon cells. Further membrane flotation assay showed that HCV NS protein was cofractionated with the FL and Y12F mutants on lipid raft fraction, but not with the mutant lacking domain of binding to actin. Furthermore, IF showed that truncated mutant was distributed in nucleus of subgenomic replicon cells. These results suggested that alpha-actinin was residing on lipid raft and may be a component of HCV replication complex.The above data indicated that alpha-actinin is residing on HCV replication site and facilitates HCV RNA replication and may require dephosporylation status. The findings may provide new insights into mechanisms of virus replication and life cycle in cells.