Bovine papillomavirus type 1 DNA replication: the transcriptional activator E2 acts in vitro as a specificity factor

Replication of the genome of human papillomaviruses (HPV) is initiated by the recruitment of the viral E1 helicase to the origin of DNA replication by the viral E2 protein, which binds specifically to the origin. We determined, for HPV type 11 (HPV-11), that the C-terminal 296 amino acids of E1 are sufficient for interaction with the transactivation domain of E2 in the yeast two-hybrid system and in vitro. This region of E1 encompasses the ATP-binding domain. Here we have examined the role of this ATP-binding domain, and of ATP, on E2-dependent binding of E1 to the origin. Several amino acid substitutions in the phosphate-binding loop (P loop), which is implicated in binding the triphosphate moiety of ATP, abolished E2 binding, indicating that the structural integrity of this domain is essential for the interaction. The structural constraints imposed on the E1 P loop may differ between HPV-11 and bovine papillomavirus type 1 (BPV-1), since the P479S substitution that inactivates BPV-1 E1 is tolerated in the HPV-11 enzyme. Other substitutions in the E1 P loop, or in two other conserved motifs of the ATP-binding domain, were tolerated, indicating that ATP binding is not essential for interaction with E2. Nevertheless, ATP-Mg stimulated the E2-dependent binding of E1 to the origin in vitro. This stimulation was maximal at the physiological temperature (37°C) and did not require ATP hydrolysis. In contrast, ATP-Mg did not stimulate the E2-dependent binding to the origin of an E1 protein containing only the C-terminal domain (353 to 649) or that of mutant E1 proteins with alterations in the DNA-binding domain. These results are discussed in light of a model in which the E1 ATP-binding domain is required for formation of the E2-binding surface and can, upon the binding of ATP, facilitate and/or stabilize the interaction of E1 with the origin.

Section: Figsupporting

confidence: 90%

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

Titolo

Pelletier

Sauvé

et al. 1999

“…E2 enhances replication of the viral DNA through complex formation with the viral E1 protein, which has characteristics of a replication initiator protein (61). In vitro and in vivo studies have demonstrated that binding of E2 to E1 increases the specificity of the origin recognition by E1 (8,20,36,39,43,54,55,63,71). The BPV1 E2 protein was first characterized as an enhancer-binding protein that could stimulate transcription from multimerized E2 binding sites located upstream of heterologous promoters in transient assays (3,27,58).…”

mentioning

confidence: 99%

Transactivation by the E2 Protein of Oncogenic Human Papillomavirus Type 31 Is Not Essential for Early and Late Viral Functions

Stubenrauch

Colbert

Laimins

1998

The activation of transcription and of DNA replication are, in some cases, mediated by the same proteins. A prime example is the E2 protein of human papillomaviruses (HPVs), which binds ACCN6GGT sequences and activates heterologous promoters from multimerized binding sites. The E2 protein also has functions in replication, where it complexes with the virally encoded origin recognition protein, E1. Much of the information on these activities is based on transient-transfection assays as well as biochemical analyses; however, their importance in the productive life cycle of oncogenic HPVs remains unclear. To determine the contributions of these E2 functions to the HPV life cycle, a genetic analysis was performed by using an organotypic tissue culture model. HPV type 31 (HPV31) genomes that contained mutations in the N terminus of E2 (amino acid 73) were constructed; these mutants retained replication activities but were transactivation defective. Following transfection of normal human keratinocytes, these mutant genomes were established as stable episomes and expressed early viral transcripts at levels similar to those of wild-type HPV31. Upon differentiation in organotypic raft cultures, the induction of late gene expression and amplification of viral DNA were detected in cell lines harboring mutant genomes. Interestingly, only a modest reduction in late gene expression was observed in the mutant lines. We conclude that the transactivation function of E2 is not essential for the viral life cycle of oncogenic HPVs, although it may act to moderately augment late expression. Our studies suggest that the primary positive role of E2 in the viral life cycle is as a replication factor.

“…In addition, E2 may prevent nucleosome formation around the ori in vivo (19). By virtue of its strong and specific affinity to the E2 protein binding site and its interaction with the E1 protein, the E2 protein is critical to the initiation of replication from ori (4,6,17,22,31,33,40), but it appears to be dispensable during elongation (20).…”

mentioning

confidence: 99%

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

Zou

Liu

Kuo

et al. 1998

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.