The E1 protein is a multifunctional, origin-binding helicase that is essential for replication of papillomaviruses. Recently, bovine papillomavirus E1 was shown to be post-translationally modified by the addition of the SUMO-1 polypeptide. Here we show that the site of sumoylation maps to lysine residue 514. This lysine and the flanking sequences are well conserved in human papillomavirus (HPV) E1 proteins. Both HPV1a and HPV18 E1 proteins are substrates for sumoylation in vitro, which is consistent with this modification being a general property of E1 proteins. Mutations, which impair the sumoylation of bovine papillomavirus E1, prevent normal nuclear accumulation of E1 with a concomitant loss of replication capacity. These results suggest that sumoylation plays a role in nuclear transport and could regulate the E1 replication function by controlling access to the nuclear replication domains. Bovine papillomavirus (BPV)1 E1 protein is the major initiator protein for viral DNA replication and thus plays a critical role in the establishment of episomal viral genomes in the host cell nucleus (1). E1 and E2 proteins are the only two viral proteins required to replicate the viral genome in vivo, whereas the rest of the replication machinery is provided by the host cell (2, 3). E1 is a DNA helicase that binds to specific sequences in the viral origin and unwinds the viral DNA for initiation of replication (4, 5). Purified E1 protein can initiate in vitro viral replication without the support of the E2 protein (6). However, the interaction of E1 with E2 protein promotes specific binding and assembly of E1 protein on the viral origin to form an active initiation complex (7,8). Additional functions of E1 include recruiting cellular replication factors, such as the DNA polymerase ␣ and replication protein A (9 -11). E1 also interacts with histone H1 (12) and SW1/SNF5 (13), suggesting that E1 plays a role in chromatin remodeling at the viral origin.Recently, we demonstrated that BPV E1 protein is posttranslationally modified by a novel process known as sumoylation (14). Sumoylation involves the covalent attachment of the small ubiquitin-related proteins (SUMO-1, -2, or -3) to target substrates (15). Sumoylation is achieved by a multistep process in a manner that is analogous to ubiquitinylation (reviewed in Ref. 16). SUMO is first activated by a heterodimeric enzyme termed Aos1/Uba2, and then the activated SUMO is covalently transferred to the SUMO-specific conjugating enzyme Ubc9 via a thioester linkage (17). Ubc9 physically interacts with various substrates and transfers SUMO to one or more lysine residues in the target protein. Unlike ubiquitinylation in which the primary function is targeting proteins for degradation, the effects of sumoylation are more substrate-specific and can result in alterations in function (18 -20), stability (21), or intracellular location (22-25) of the modified protein. For BPV E1, a mutant unable to interact with Ubc9 showed altered intranuclear distribution (14). As BPV E1 is the first kno...
The E1 protein of bovine papillomavirus (BPV) is a site-specific DNA binding protein that recognizes an 18-bp inverted repeat element in the viral origin of replication. Sequence-specific DNA binding function maps to the region from approximately amino acids 140 to 300, and isolated polypeptides containing this region have been shown to retain origin binding in vitro. To investigate the sequence and structural characteristics which contribute to sequence-specific binding, the primary sequence of this region was examined for conserved features. The BPV E1 DNA binding domain (E1DBD) contains three major hydrophilic domains (HR1, amino acids 179-191; HR2, amino acids 218 to 230; and HR3, amino acids 241 to 252), of which only HR1 and HR3 are conserved among papillomavirus E1 proteins. E1DBD proteins with lysine-to-alanine mutations in HR1 and HR3 were severely impaired for DNA binding function in vitro, while a lysine-to-alanine mutation in HR2 had a minimal effect on DNA binding. Mutation of adjacent threonine residues in HR1 (T187 and T188) revealed that these two amino acids made drastically different contributions to DNA binding, with the T187 mutant being severely defective for origin binding whereas the T188 mutant was only mildly affected. Helical wheel projections of HR1 predict that T187 is on the same helical face as the critical lysine residues whereas T188 is on the opposing face, which is consistent with their respective contributions to DNA binding activity. To examine E1 binding in vivo, a yeast one-hybrid system was developed. Both full-length E1 and the E1DBD polypeptide were capable of specifically interacting with the E1 binding site in the context of the yeast genome, and HR1 was also critical for this in vivo interaction. Overall, our results indicate that HR1 is essential for origin binding by E1, and the features and properties of HR1 suggest that it may be part of a recognition sequence that mediates specific E1-nucleotide contacts.Bovine papillomavirus (BPV) replication requires only two viral proteins, E1 and E2 (40), with the rest of the replication machinery supplied by the host cell (27). Both the E1 and E2 proteins are site-specific DNA binding proteins which recognize sequences in the viral origin of replication (1,41,42). The E2 binding site (E2BS) is a 12-bp partial palindrome (1, 22), while E1 binds to an 18-bp imperfect inverted repeat sequence (18,19,34). In vitro, only the E1 protein is absolutely required, indicating that the E2 protein does not supply a requisite replication function (3, 5). Instead, E2 appears to act as an auxiliary factor that interacts directly with E1 (24, 35, 44) and increases binding site specificity (5, 32). Moreover, at low protein concentrations, E2-E1 complexes facilitate loading and assembly of additional E1 molecules on the origin to form the active initiation complex (30). Initial binding of E1 induces distortion in the origin DNA that is a likely prelude to origin unwinding, and this distortion of the origin DNA is also enhanced by E2 (13, 30). U...
The E1 proteins are the essential origin recognition proteins for papillomavirus (PV) replication. E1 proteins bind to specific DNA elements in the viral origin of replication and assemble into hexameric helicases with the aid of a second viral protein, E2. The resultant helicase complex initiates origin DNA unwinding to provide the template for subsequent syntheses of progeny DNA. In addition to ATP-dependent helicase activity, E1 proteins interact with and recruit several host cell replication proteins to viral origin, including DNA polymerase alpha and RPA. This review will compare the basic structures and features of the human (HPV) and bovine (BPV1) papillomaviruses with an emphasis on mechanisms of replication function.
The interaction between papillomavirus E1 and E2 proteins is essential for viral genome replication. Using both in vivo and in vitro assays to evaluate the regions of the two proteins necessary for the E1-E2 interaction, three independent interactions were identified for bovine papillomavirus E1: the N terminus of E1 (E1N, residues 1-311) interacts with the E2 transactivation domain (E2TAD) and the E2 DNA-binding domain (E2DBD) and the C terminus of E1 (E1C, residues 315-605) interacts with E2. Nine mutations within E1N were evaluated for their effects on E2 interaction. Five mutations eliminated interaction with the E2TAD ; four of these were located within two previously identified conserved, hydrophilic regions, HR1 and HR3. Since HR1 and HR3 residues appear to comprise the origin of replication recognition element for E1, simultaneous interaction with the E2TAD during initiation complex formation would seem unlikely. Consistent with this inference is the fact that three of the five mutants defective for E2TAD binding exhibited wild-type levels of replication. The replication-positive phenotype of these mutants suggests that the E1N-E2TAD interaction is not essential for replication function and is probably involved in some other E1-E2 function, such as regulating transcription. Only one of the five mutations defective for E2TAD binding also prevented E2DBD interaction, indicating that the regions of E1N that interact with the E2TAD and the E2DBD are not identical. The ability of E1N to cooperatively interact with E2 bound to E2-binding site (E2BS) 11 versus E2BS12 was also examined, and cooperative binding was only observed when E2 was bound to E2BS12.
Bovine papillomavirus type 1 (BPV-1) requires viral proteins E1 and E2 for efficient DNA replication in host cells. E1 functions at the BPV origin as an ATP-dependent helicase during replication initiation. Previously, we used alanine mutagenesis to identify two hydrophilic regions of the E1 DNA binding domain (E1DBD), HR1 (E1 [179][180][181][182][183][184][185][186][187][188][189][190][191] ) and HR3 (E1 241-252 ), which are critical for sequence-specific recognition of the papillomavirus origin. Based on sequence and structure, these regions are similar in spacing and location to DNA binding regions A and B2 of T antigen, the DNA replication initiator of simian virus 40 (SV40). HR1 and A are both part of extended loops which are supported by residues from the HR3 and B2 ␣-helices. Both elements contain basic residues which may contact DNA, although lack of cocrystal structures for both E1 and T antigen make this uncertain. To better understand how E1 interacts with origin DNA, we used random mutagenesis and a yeast one-hybrid screen to select mutations of the E1DBD which disrupt sequence-specific DNA interactions. From the screen we selected seven single point mutants and one double point mutant (F175S, N184Y/K288R, D185G, V193M, F237L, K241E, R243K, and V246D) for in vitro analysis. All mutants tested in electrophoretic mobility shift assays displayed reduced sequence-specific DNA binding compared to the wild-type E1DBD. Mutants D185G, F237L, and R243K were rescued in vitro for DNA binding by the replication enhancer protein E2. We also tested the eight mutations in full-length E1 for the ability to support DNA replication in Chinese hamster ovary cells. Only mutants D185G, F237L, and R243K supported significant DNA replication in vivo which highlights the importance of E1DBD-E2 interactions for papillomavirus DNA replication. Based on the specific point mutations examined, we also assigned putative roles to individual residues in DNA binding. Finally, we discuss sequence and spacing similarities between E1 HR1 and HR3 and short regions of two other DNA tumor virus origin-binding proteins, SV40 T antigen and Epstein-Barr virus nuclear antigen 1 (EBNA1). We propose that all three proteins use a similar DNA recognition mechanism consisting of a loop structure which makes base-specific contacts (HR1) and a helix which primarily contacts the DNA backbone (HR3).Papillomaviruses are members of the papovavirus family and comprise both human and animal strains. In addition to causing common cutaneous warts, papillomaviruses can induce various skin and mucosal lesions (35,36,39). Some of these lesions may progress to malignant carcinomas, depending on the particular viral strain involved as well as environmental factors. Malignancies which are highly associated with papillomavirus infection include laryngeal, cervical, and other anogenital cancers, and it is noteworthy that human papillomavirus (HPV) types 16, 18, 31, 33, and 45 pose an especially high risk for females, as they are estimated to be present in at least ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.