Molecular mechanism of initiation of human papillomavirus (HPV) DNA replication

Abstract

Human papillomaviruses (HPVs) are small double-stranded DNA viruses responsible for a significant burden of disease, including cervical, oropharyngeal and other anogenital cancers. The initiation of HPV DNA replication depends on the cooperative function of the viral E1 helicase and the E2 initiation protein. E1 assembles as a hexameric helicase that unwinds the viral genome, while E2 facilitates E1 recruitment to the replication origin and modulates its enzymatic functions. In this study, full-length HPV16 E1 helicase was successfully expressed and purified to homogeneity. Biochemical analyses confirmed that the purified protein forms oligomeric complexes consistent with its helicase function and exhibits ATPase activity that is significantly stimulated in the presence of single-stranded DNA. Helicase assays demonstrated DNA unwinding activity dependent on ATP hydrolysis. ☐ Electrophoretic mobility shift assays revealed that stable E1–E2–DNA complexes require the presence of E2, while the putative E1 binding site within the viral origin is dispensable, for this complex assembly. E2 protein facilitated the E1 recruitment, indicating that E2 plays a primary role in complex assembly. Furthermore, E2 was observed to inhibit both the ATPase and helicase activities of E1, suggesting a regulatory mechanism that controls the origin unwinding. ☐ Quantitative characterization of the E1–E2 interaction was performed using bio-layer interferometry. The binding studies demonstrated that E1 and E2 form a high-affinity complex with nanomolar dissociation constants. The presence of ATP or ADP promoted dissociation of the complex, whereas single-stranded DNA had minimal impact on nucleotide-induced dissociation. Additional experiments showed that critical glutamic acid residues in E2 (E20 and E39) severely impaired E1 binding, confirming their importance in maintaining complex stability. ☐ These findings provide new insights into the mechanistic basis of HPV DNA replication initiation. The work establishes that E2 is both an essential factor for recruiting E1 to the origin and a negative regulator of its enzymatic activities. Moreover, the results highlight the dynamic modulation of E1–E2 interactions by nucleotide cofactors. Together, this study advances the understanding of papillomavirus replication and offers a foundation for future efforts aimed at targeting viral replication processes therapeutically.