Identification of Domains of the Human Papillomavirus Type 11 E1 Helicase Involved in Oligomerization and Binding to the Viral Origin

Abstract

The E1 helicase of papillomavirus is required, in addition to host cell DNA replication factors, during the initiation and elongation phases of viral episome replication. During initiation, the viral E2 protein promotes the assembly of enzymatically active multimeric E1 complexes at the viral origin of DNA replication. In this study we used the two-hybrid system and chemical cross-linking to demonstrate that human papillomavirus type 11 (HPV11) E1 can self-associate in yeast and form hexamers in vitro in a reaction stimulated by singlestranded DNA. Self-association in yeast was most readily detected using constructs spanning the E1 C-terminal domain (amino acids 353 to 649) and was dependent on a minimal E1-E1 interaction region located between amino acids 353 and 431. The E1 C-terminal domain was also able to oligomerize in vitro but, in contrast to wild-type E1, did so efficiently in the absence of single-stranded DNA. Sequences located between amino acids 191 and 353 were necessary for single-stranded DNA to modulate oligomerization of E1 and were also required, together with the rest of the C terminus, for binding of E1 to the origin. Two regions within the C-terminal domain were identified as important for oligomerization: the ATP-binding domain and region A, which is located within the minimal E1-E1 interaction domain and is one of four regions of E1 that is highly conserved with the large T antigens of simian virus 40 and polyomavirus. Amino acid substitutions of highly conserved residues within the ATP-binding domain and region A were identified that reduced the ability of E1 to oligomerize and bind to the origin in vitro and to support transient DNA replication in vivo. These results support the notion that oligomerization of E1 occurs primarily through the C-terminal domain of the protein and is allosterically regulated by DNA and ATP. The bipartite organization of the E1 C-terminal domain is reminiscent of that found in other hexameric proteins and suggests that these proteins may oligomerize by a similar mechanism.Papillomaviruses are small DNA viruses that are the etiological agents of benign and malignant lesions of the differentiating mucosal and cutaneous epithelium (reviewed in references 6, 20, 45, and 61). The life cycle of these viruses is closely associated with the differentiation program that occurs in the epithelium. Soon after infection, the viral genome is established as a low-copy-number extrachromosomal episome in the nuclei of infected basal cells. As these infected cells start to differentiate from the basal layer toward the upper portion of the epithelium, the viral episome is replicated and maintained at approximately 50 copies per cell. When the infected cells reach the suprabasal layers, amplification of the viral episome occurs to a high copy number, probably as a result of a change in the mode of DNA replication from a bidirectional theta mode to a rolling-circle mode (11). In these upper layers, capsid proteins are synthesized and viral particles are assembled, and they are e...

On helicases and other motor proteins

“…In the case of T7gp4, oligomerization is mediated by an extended 'tail' that appends the primase domain and sits on the adjacent subunit [36 ]. Oligomerization of DnaB, the SF3 helicases [46], and MCM proteins [47] appears to derive from a second domain that is static and also forms tight and extensive interactions with adjacent subunits through apparently inflexible interfaces. These properties classify this region as a 'collar' similar to that described earlier in the case of clamp-loaders [48,49].…”

ATP-Dependent Minor Groove Recognition of TA Base Pairs Is Required for Template Melting by the E1 Initiator Protein

“…Viral initiator proteins such as the simian virus 40 (SV40) large T antigen (T-Ag) and the papillomavirus E1 protein have long been known to melt their respective origins of DNA replication, as detected by oxidation with KMnO 4 (4,5,14,22,24,25). However, little information exists about which forms of these proteins execute melting, which parts of the polypeptide are responsible for the melting activity, and whether this process is DNA sequence dependent (5, 27).The E1 proteins from papillomaviruses are ϳ70-kDa polypeptides which, in addition to DNA melting activity, have DNA helicase activity (19,20,30,33,35,37,38,40) and also bind DNA. DNA binding by E1 is the result of two different DNA binding activities.…”

“…The E1 proteins from papillomaviruses are ϳ70-kDa polypeptides which, in addition to DNA melting activity, have DNA helicase activity (19,20,30,33,35,37,38,40) and also bind DNA. DNA binding by E1 is the result of two different DNA binding activities.…”

The Fidelity of HPV16 E1/E2-mediated DNA Replication

“…E2 can then regulate transcription from the adjacent promoter that controls the expression of the HPV oncogenes E6 and E7; E2 can either activate or repress transcription depending upon E2 protein levels and the cell type (5, 6). As well as regulating transcription E2 is also essential for replication of the viral genome; there are three E2 DNA binding sequences surrounding the viral origin (ori) of replication and a physical interaction between E2 and E1 results in recruitment of E1 to the ori sequence (7,8,9). Following recruitment by E2 the E1 protein interacts with the ori DNA sequence and then forms a hexameric complex required for replication of the viral genome by virtue of its helicase activity (10).…”