Gallus Heat shock cognate protein 70, a novel binding partner of Apoptin

Chicken anemia virus (CAV) infection of newly hatched chickens induces generalized lymphoid atrophy and causes immunosuppressive. VP3, also known as Apoptin, is non-structural protein of CAV. Apoptin specifically induces apoptosis in transformed or tumor cells but not in normal cells. In particular, there are no known cellular homologues of Apoptin hindering genetic approaches to elucidate its cellular function. Although a number of Apoptin-interacting molecules have been identified, the molecular mechanism underlying Apoptin's action is still poorly understood. To learn more about the molecular mechanism of Apoptin's action, we searched for Apoptin associated proteins.

We identified Gallus Hsc70 as an Apoptin binding protein and showed the translocation of Hsc70 into the nuclei of DF-1 cells treated with Apoptin. Hsc70 regulates RelA/p65 gene expression induced by Apoptin.

Using yeast two-hybrid and colony-life filter approaches we got five positive yeast clones. Through sequencing and BLASTed against NCBI, one of the clones was confirmed containing Gallus heat shock cognate protein 70 (Hsc70). Hsc70 gene was clone into pRK5-Flag plasmid, coimmunoprecipitation assay show both exogenous Hsc70 and endogenous Hsc70 can interact with Apoptin. Truncated Apoptin expression plasmids were made and coimmunoprecipitation were performed, the results show the binding domain of Apoptin with Hsc70 is located between amino acids 30-60. Truncated expression plasmids of Hsc70 were also constructed and coimmunoprecipitation were performed, the results show the peptide-binding and variable domains of Hsc70 are responsible for the binding to Apoptin. Confocal assays were performed and results show that under physiological condition Hsc70 is predominantly distributed in cytoplasm, whereas Hsc70 is translocated into the nuclei and colocalized with Apoptin in the presence of Apoptin in DF-1 cell. Functional studies show that Apoptin markedly down-regulate the mRNA level of RelA/p65 in DF-1 cell. To explore the effect of Hsc70 on Apoptin-mediated RelA/p65 gene expression, we have searched two Hsc70 RNAi sequences, and found that all of them dramatically inhibited the expression of endogenous Hsc70, with the #2 Hsc70 RNAi sequence being the most effective. Knockdown of Hsc70 show Apoptin-inhibited RelA/p65 expression was abolished. Our data demonstrate that Hsc70 is responsible for the down-regulation of Apoptin induced RelA/p65 gene expression.