DNA-binding domain of bovine papillomavirus type 1 E1 helicase: structural and functional aspects

Abstract

The El protein of bovine papillomavirus type 1 is a multifunctional enzyme required for papillomaviral DNA replication. It assists in the initiation of replication both as a site-specific DNA-binding protein and as a DNA helicase. Previous work has indicated that at limiting El concentrations, the E2 protein is required for efficient El binding to the replication origin. In this study, we have defined the domain of the El protein required for site-specific DNA binding. Experiments with a series of truncated proteins have shown that the first amino-terminal 299 amino acids contain the DNA-binding domain; however, the coterminal M protein, which is homologous to El for the first 129 amino acids, does not bind origin DNA. A series of small internal deletions and substitution mutations in the DNA-binding domain of El show that specific basic residues in this region of the protein, which are conserved in all El proteins of the papillomavirus family, likely play a direct role in binding DNA and that a flanking conserved hydrophobic subdomain is also important for DNA binding. A region of El that interacts with E2 for cooperative DNA binding is also retained in carboxy-terminal truncated proteins, and we show that the ability of full-length El to complex with E2 is sensitive to cold. The El substitution mutant proteins were expressed from mammalian expression vectors to ascertain whether site-specific DNA binding by El is required for transient DNA replication in the cell. These El proteins display a range of mutant phenotypes, consistent with the suggestion that site-specific binding by El is important. Interestingly, one El mutant which is defective for origin binding but can be rescued for such activity by E2 supports significant replication in the cell.

Isolation of an amino-terminal region of bovine papillomavirus type 1 E1 protein that retains origin binding and E2 interaction capacity

“…The carboxy-terminal boundary that we determined is consistent with the previous observation that an E1 1-299 protein retained origin binding activity (15) but is in contrast to the recent results of Sarafi and McBride that an E1 1-328 protein lacked origin binding activity (12). Furthermore, Sarafi and McBride showed that an E1 1-378 and an E1 162-422 protein bound the origin but that an E1 162-378 protein did not (12).…”

“…In vitro, E1 alone is sufficient to initiate replication (3), but in vivo the viral full-length E2 transcriptional activator protein is also absolutely required (16). E1 can form complexes with the E2 protein (2,8,15,20), and both binding to and unwinding of origin DNA by E1 is enhanced by these cooperative interactions with E2 (4,9,13,20). Consequently, the replication function of E1 appears dependent upon both protein-DNA and protein-protein contacts.…”

“…Definition of the regions of E1 involved in each of these activities will facilitate our understanding of how protein-DNA complexes assemble to mediate replication initiation. Previous studies have begun the mapping of both the DNA binding domain (12,15) and the E2 interaction domain(s) of E1 (1,8,12,15) but have yielded partially conflicting results. We have now refined the mapping of the DNA binding domain and further demonstrate that this isolated domain is capable of both binding origin DNA and interacting with the E2 protein.…”

The Interaction between Human Papillomavirus Type 16 El and E2 Proteins is Blocked by An Antibody to the N‐Terminal Region of E2

“…The binding site of E2 on the E l molecule has been mapped to amino acids 384-516 (Lusky and Fontane, 1991) and amino acids 458-605 (Thorner et al, 1993) of BPV1. In HPV11, preliminary data localised the site of complex formation to a region of the E l protein before amino acid 251 (Bream et al, 1993).…”

The initiator protein E1 binds to the bovine papillomavirus origin of replication as a trimeric ring-like structure.

“…The bovine papillomavirus (BPV) genome encodes two proteins that are essential for viral DNA replication . The El protein is a 72 kDa nuclear protein with a sequence-specific DNA binding activity that serves to recognize the ori, has DNA-dependent ATPase activity and can function as a DNA helicase Wilson and Ludes-Meyers, 1991;Yang et al, 1991;Holt et al, 1993;Seo et al, 1993a; ( Oxford University Press Thorner et al, 1993;Yang et al, 1993). The E2 protein functions as a sequence-specific activator of transcription (Spalholz et al, 1985;Androphy et al, 1987;McBride et al, 1991), but is also required for viral DNA replication.…”