P-E2 An Hsc70-binding peptide derived from SV40 Large T antigen is a potent inhibitor of polyomavirus DNA replication

Abstract

The Large T Antigen (TAg) of polyomaviruses (PyVs) is a helicase required for viral DNA replication and an oncoprotein that stimulates quiescent cells to reenter a proliferative state conducive to viral DNA synthesis. In doing so, TAg engages in multiple protein-protein interactions with host DNA replication and cell-cycle control machineries. To identify which viral-host interaction might be susceptible to competition by a peptidic inhibitor, we scanned the TAg protein with 50-60 amino acid long overlapping peptides for functional dominant-negative inhibitors in our cell-based assay of SV40 DNA replication. This method identified a potent inhibitory peptide derived from the first exon of TAg which bears high sequence homology with the J-domain of the Hsp40/DNAJ co-chaperone family, and is required for viral DNA replication. Studies have found that the J-domain interacts with Hsc70 and stimulates its ATPase/chaperone activity, to release E2F from pRb-containing complexes bound to TAg to promote viral DNA replication and transformation. Characterization of our J-domain inhibitory peptide (aa 2-64) confirmed that it interacts with Hsc70. Mutagenesis of the HPD motif conserved in all known DnaJ homologues, abolished the interaction of the peptide with Hsc70 and its ability to inhibit SV40 DNA replication. Notably, the overexpression of this J-domain peptide could also inhibit viral DNA replication for the clinically relevant human PyVs JCV, BKV and MCV, whose reactivation in immunocompromised patients is associated with the development of progressive multifocal leukencephalopathy, BK-induced nephropathy, and Merkel Cell Carcinoma respectively. Collectively, these results suggest that the J-domain peptide inhibits viral DNA replication by competing with full length TAg for Hsc70 binding. As such, this peptide represents a promising tool to study the function of Hsc70 in PyV DNA replication and to develop therapies for the treatment of PyV-associated diseases including Merkel Cell Carcinoma caused by MCPyV.