The Carboxyl-Terminal Region of the Human Papillomavirus Type 16 E1 Protein Determines E2 Protein Specificity during DNA Replication

Abstract

The mechanism of DNA replication is conserved among papillomaviruses. The virus-encoded E1 and E2 proteins collaborate to target the origin and recruit host DNA replication proteins. Expression vectors of E1 and E2 proteins support homologous and heterologous papillomaviral origin replication in transiently transfected cells. Viral proteins from different genotypes can also collaborate, albeit with different efficiencies, indicating a certain degree of specificity in E1-E2 interactions. We report that, in the assays of our study, the human papillomavirus type 11 (HPV-11) E1 protein functioned with the HPV-16 E2 protein, whereas the HPV-16 E1 protein exhibited no detectable activity with the HPV-11 E2 protein. Taking advantage of this distinction, we used chimeric E1 proteins to delineate the E1 protein domains responsible for this specificity. Hybrids containing HPV-16 E1 amino-terminal residues up to residue 365 efficiently replicated either viral origin in the presence of either E2 protein. The reciprocal hybrids containing amino-terminal HPV-11 sequences exhibited a high activity with HPV-16 E2 but no activity with HPV-11 E2. Reciprocal hybrid proteins with the carboxyl-terminal 44 residues from either E1 had an intermediate property, but both collaborated more efficiently with HPV-16 E2 than with HPV-11 E2. In contrast, chimeras with a junction in the putative ATPase domain showed little or no activity with either E2 protein. We conclude that the E1 protein consists of distinct structural and functional domains, with the carboxyl-terminal 284 residues of the HPV-16 E1 protein being the primary determinant for E2 specificity during replication, and that chimeric exchanges in or bordering the ATPase domain inactivate the protein.

Human Papillomavirus DNA Replication Compartments in a Transient DNA Replication System

“…For HPV, the E2 protein is the primary ori recognition protein, as the E2BS is essential but the E1BS is not (8,29,34,35,65). Furthermore, certain combinations of E1 and E2 proteins encoded by heterologous virus types support ori replication with a reduced efficiency or not at all (4,9,79). A direct interaction between pairs of E1 and E2 proteins of the same HPV types or BPV type 1 (BPV-1) has indeed been demonstrated (20,24,43,44,52).…”

“…However, the specificity and efficiency of replication are significantly enhanced by collaboration with the E2 protein (29,76). In contrast, in transfected cells, both E1 and E2 are required for ori replication, with few exceptions (4,9,54,79). The reasons for the difference in the requirements for E2 between the two replication systems are not completely understood.…”

Role of the ATP-Binding Domain of the Human Papillomavirus Type 11 E1 Helicase in E2-Dependent Binding to the Origin

“…These findings also indicate that we must exert caution in interpreting the effect of substitutions in the P loop of E1, and perhaps also in that of other NTP-binding proteins, and not assume that these substitutions only affect NTP binding and hydrolysis. Finally, the notion that the precise geometry of the E1 ATP-binding domain is critical for function is also supported by the recent finding that chimeric E1 proteins, in which the junction between HPV-11 and HPV-16 sequences was within the ATPase domain, were unable to support HPV DNA replication (66).…”

“…For BPV-1, an E2binding region was identified between amino acids 200 and 605 of E1, but a slightly larger domain (165 to 605) was required for cooperative binding with E2 at the origin (45). Point mutations and deletions in the BPV-1 E1 C-terminal domain (31), as well as the study of HPV-11-HPV-16 chimeric E1 proteins (66), also indicated a requirement for this region in interaction with E2.…”

E1 and E2 Viral Proteins as Therapeutic Targets for Development of Antiviral Agents

“…HPV DNA replication initiates with the cooperative join of E1 and E2 with specific DNA sequences inside the viral origin [15][16][17]. Formation of this ternary compound E1-E2-ori depends on the interaction between both proteins with DNA, but also there is a critical interaction between the E2 transactivation domain of N-terminal (TAD) and the E1 enzymatic domain of E1 C-terminal [25]. Assembly of this initial complex E1-E2-ori is the starting point for additional E1 molecule recruitment [26,27] and its assembly in hexamers and double hexamers that display activity in ATPase and helicase [28,29].…”