The bovine papillomavirus E2 protein modulates the assembly of but is not stably maintained in a replication-competent multimeric E1-replication origin complex.

Abstract

Initiation of bovine papillomavirus (BPV) DNA synthesis in vivo and in vitro depends on the interaction of the viral initiator protein El with the replication origin (ori+ DNA). The viral E2 protein assists this interaction, resulting in a cooperative assembly of both proteins on the replication origin. Using gel mobility-shift experiments, we demonstrate that in the presence of both El and E2 proteins two classes of ori+ DNA complexes were formed: complex 1 (cl) and complex 2 (c2). Formation of ci depended on both the El and E2 proteins and both proteins were contained within ci. The generation of c2 was dependent on the El protein and could be enhanced by E2, but the E2 protein was not detected within c2. At high E2/El ratios, ci was the dominant complex formed. Under these conditions, El-dependent BPV DNA synthesis in vitro was inhibited. At low E2/El ratios, the stimulation of c2 was correlated with the stimulation of BPV DNA replication by E2 in vitro. These data suggest that E2 assists El in the formation of an intermediate ci complex, which is replication inactive. The ci complex is converted in turn to the replicationactive c2 complex, which contains El but lacks E2. We propose that the ratios of ci and c2 formed in response to the levels of El and E2 proteins determine the potential for BPV DNA synthesis in vitro and in vivo and may contribute to copy number regulation of BPV plasmids within the cell.Studies of prokaryotic replication systems and small DNA viruses in eukaryotic cells have shown that initiation of DNA synthesis requires the interaction of a specific initiator protein with its cognate replication origin (see ref. 1 for review). In addition, initiator proteins such as the simian virus 40 large tumor antigen facilitate the recruitment of cellular proteins and DNA polymerases to generate an active replication complex at the origin (2-4).Papillomaviruses, such as bovine papillomaviruses (BPV), are maintained in latently infected cells as nuclear plasmids (see ref. 5 for review) and thus provide an additional eukaryotic system to investigate the assembly of a replication complex and to study the control of copy number.BPV DNA replication in vivo requires two viral proteinsthe 68-kDa El and the 48-kDa E2 polypeptides (6). The minimal in vivo origin see also Fig. 5) contains an A+T-rich region (ATR), an El binding site (BS) that includes a region of dyad symmetry (DSR), and the E2 BS12 (nt 16-27). El-dependent BPV DNA synthesis in vitro does not require the E2 protein at high levels of El (9-11). At low levels of El, E2 enhances in vitro DNA synthesis (9) even in the absence of a well-defined E2 BS (12).The El polypeptide specifically binds to the BPV replication origin (ori+) (7, 9, 13) and this process can be stimulated and stabilized in the presence ofATP (14, 15). In addition, the El-associated DNA-dependent NTPase and NTP-dependent DNA helicase activities lead to the specific unwinding of BPV origin-containing plasmid DNAs (10,14).In support of a direct role for the 48-kDa E2 tra...

Human Papillomavirus DNA Replication

“…The elution profile of the p180⅐E1 protein complex suggests that the interaction between E1 and p180 is not stoichiometric and perhaps more than one molecule of E1 interacts with one molecule of p180, in agreement with previous observations that E1 can exist as a multimer in solution (14,20,21,23). Finding that only a portion of the p180 associated with E1 during gel filtration and a small percentage of the total polymerase ␣ activity co-immunoprecipitated from lysates of co-infected insect cells (data not shown) leads us to the hypothesis that E1 may exist as a multimer in solution.…”

“…As a result, high concentrations of E1 can bind DNA nonspecifically and initiate ori-independent replication at low efficiency in vitro (3, 4). It has been proposed that the replication competent form of BPV-1 E1 is a multimeric complex of 10 -12 E1 molecules (20). Recently, the HPV-11 E1 protein has been shown to bind to the human chaperone protein Hsp40, and in its presence, a dihexameric E1 complex forms on the ori (23), mirroring the structure of SV40 T antigen on the SV40 ori (24) as well as other known Escherichia coli helicases (25).…”

“…Therefore, the addition of E2 protein to a cell-free replication assay enhances ori-dependent and suppresses ori-independent replication. Based on these data the following model of E2/E1 interaction during initiation of bovine papillomaviral DNA replication has been proposed (20). Once the first molecule of E1 is loaded onto the origin by E2, E2 is then released from the origin, allowing E1 to multimerize into a replication-competent form.…”

“…It forms a complex with E1 (35), and this E1⅐E2 complex has increased sequence specificity for binding to the E1 and E2 cognate sites in the viral origin (36,37). Thus one of the critical functions of E2 in viral DNA replication is to interact with and recruit E1 to the viral origin of replication by virtue of the stronger DNA binding affinity and specificity for E2 (20,(35)(36)(37)(38)(39)(40)(41). Therefore, the addition of E2 protein to a cell-free replication assay enhances ori-dependent and suppresses ori-independent replication.…”

The X-ray structure of the papillomavirus helicase in complex with its molecular matchmaker E2

“…How E2 regulates E1 assembly in this conversion process, as well as its ultimate fate, is not clear. For BPV1, following the binding of additional E1 molecules, E2 is displaced from the viral origin (Lusky et al 1994;Sanders and Stenlund 1998). For HPV-11, it has been reported that E2 remains associated with the DNA during the assembly of E1 molecules, even though E2 might serve as a potential roadblock for extensive unwinding (Lin et al 2002).…”