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

The E1 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 E1 concentrations, the E2 protein is required for efficient E1 binding to the replication origin. In this study, we have defined the domain of the E1 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 E1 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 E1 show that specific basic residues in this region of the protein, which are conserved in all E1 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 E1 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 E1 to complex with E2 is sensitive to cold. The E1 substitution mutant proteins were expressed from mammalian expression vectors to ascertain whether site-specific DNA binding by E1 is required for transient DNA replication in the cell. These E1 proteins display a range of mutant phenotypes, consistent with the suggestion that site-specific binding by E1 is important. Interestingly, one E1 mutant which is defective for origin binding but can be rescued for such activity by E2 supports significant replication in the cell.     

The E1 replication protein of bovine papillomavirus type 1 contains an extended nuclear localization signal that includes a p34cdc2 phosphorylation site.

Bovine papillomavirus (BPV) DNA replication occurs in the nucleus of infected cells. Most enzymatic activities are carried out by host cell proteins, with the viral E1 and E2 proteins required for the assembly of an initiation complex at the replication origin. In latently infected cells, viral DNA replication occurs in synchrony with the host cell chromosomes, maintaining a constant average copy number of BPV genomes per infected cell. By analyzing a series of mutants of the amino-terminal region of the E1 protein, we have identified the signal for transport of this protein to the cell nucleus. The E1 nuclear transport motif is highly conserved in the animal and human papillomaviruses and is encoded in a similar region in the related E1 genes. The signal is extended relative to the simple nuclear localization signals and contains two short amino acid sequences which contribute to nuclear transport, located between amino acids 85 and 108 of the BPV-1 E1 protein. Mutations in either basic region reduce nuclear transport of E1 protein and interfere with viral DNA replication. Mutations in both sequences simultaneously prevent any observable accumulation of the protein and reduce replication in transient assays to barely detectable levels. Surprisingly, these mutations had no effect on the ability of viral genomes to morphologically transform cells, although the plasmid DNA in the transformed cells was maintained at a very low copy number. Between these two basic amino acid blocks in the nuclear transport signal, at threonine 102, is a putative site for phosphorylation by the cell cycle regulated kinase p34cdc2. Utilizing an E1 protein purified from either a baculovirus vector system or Escherichia coli, we have shown that the E1 protein is a substrate for this kinase. An E1 gene mutant at threonine 102 encodes for a protein which is no longer a substrate for the p34cdc2 kinase. Mutation of this threonine to isoleucine had no observable effect on either nuclear localization of E1 or DNA replication of the intact viral genome.     

Identification of a short, hydrophilic amino acid sequence critical for origin recognition by the bovine papillomavirus E1 protein

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.     

DNA binding specificity of the bovine papillomavirus E1 protein is determined by sequences contained within an 18-base-pair inverted repeat element at the origin of replication.

Bovine papillomavirus type 1 (BPV-1) DNA replicates episomally and requires two virally expressed proteins, E1 and E2, for this process. Both proteins bind to the BPV-1 genome in the region that functions as the origin of replication. The binding sequences for the E2 protein have been characterized previously, but little is known about critical sequence requirements for E1 binding. Using a bacterially expressed E1 fusion protein, we examined binding of the BPV-1 E1 protein to the origin region. E1 strongly protected a 28-bp segment of the origin (nucleotides 7932 to 15) from both DNase I and exonuclease III digestion. Additional exonuclease III protection was observed beyond the core region on both the 5' and 3' sides, suggesting that E1 interacted with more distal sequences as well. Within the 28-bp protected core, there were two overlapping imperfect inverted repeats (IR), one of 27 bp and one of 18 bp. We show that sequences within the smaller, 18-bp IR element were sufficient for specific recognition of DNA by E1 and that additional BPV-1 sequences beyond the 18-bp IR element did not significantly increase origin binding by E1 protein. While the 18-bp IR element contained sequences sufficient for specific binding by E1, E1 did not form a stable complex with just the isolated 18-bp element. Formation of a detectable E1-DNA complex required that the 18-bp IR be flanked by additional DNA sequences. Furthermore, binding of E1 to DNA containing the 18-bp IR increased as a function of overall increasing fragment length. We conclude that E1-DNA interactions outside the boundaries of the 18-bp IR are important for thermodynamic stabilization of the E1-DNA complex. However, since the flanking sequences need not be derived from BPV-1, these distal E1-DNA interactions are not sequence specific. Comparison of the 18-bp IR from BPV-1 with the corresponding region from other papillomaviruses revealed a symmetric conserved consensus sequence, T-RY--TTAA--RY-A, that may reflect the specific nucleotides critical for E1-DNA recognition.     

Functional Mapping of the DNA Binding Domain of Bovine Papillomavirus E1 Protein

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(179-191)) 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 alpha-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).     

Bovine papillomavirus E1 protein is sumoylated by the host cell Ubc9 protein

Papillomavirus E1 protein is the replication initiator that recognizes and binds to the viral origin and initiates DNA strand separation through its ATP-dependent helicase activity. The E1 protein also functions in viral DNA replication by recruiting several cellular proteins to the origin, including host DNA polymerase alpha and replication protein A. To identify other cellular proteins that interact with bovine papillomavirus E1, an HeLa cDNA library was screened using a yeast two-hybrid assay. The host cell sumoylating enzyme, Ubc9, was found to interact specifically with E1 both in vitro and in vivo. Mapping studies localized critical E1 sequences for interaction to amino acids 315-459 and strongly implicated leucine 420 as critical for E1.Ubc9 complex formation. In addition to binding E1, Ubc9 catalyzed the covalent linkage of the ubiquitin-like protein, SUMO-1, to E1. An E1 mutant unable to bind Ubc9 showed normal intracellular stability, but was impaired for intranuclear distribution. Failure to accumulate in appropriate nuclear subdomains may account for the previously demonstrated replication defect of a human papillomavirus 16 E1 protein that was also unable to bind Ubc9 and suggests that sumoylation is a functionally important modification with regulatory implications for papillomavirus replication.     

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

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.     

Binding of the E1 and E2 proteins to the origin of replication of bovine papillomavirus.

DNA replication of bovine papillomavirus (BPV) requires two viral proteins encoded from the E1 and E2 open reading frames. E1 and E2 are sequence-specific DNA binding proteins that bind to their cognate binding sites in the BPV origin of replication (ori). The E1 and E2 proteins can interact physically with each other, and this interaction results in cooperative binding when binding sites for both proteins are present. We have analyzed the binding of E1 to the ori in the absence and presence of E2, using DNase I footprint analysis, gel mobility shift assays, and interference analysis. We have also generated a large number of point mutations in the E1 binding site and tested them for binding of E1 as well as for activity in DNA replication. Our results demonstrate that E1 binds to the ori in different forms in the absence and presence of E2 and that E2 has both a quantitative and a qualitative effect on the binding of E1. Our results also suggest that the ori contains multiple overlapping individual E1 recognition sequences which together constitute the E1 binding site and that different subsets of these recognition sequences are used for binding of E1 in the presence and absence of E2.     

Functional analysis of a carboxyl-terminal phosphorylation mutant of the bovine papillomavirus E1 protein

The papillomavirus E1 protein is essential for viral DNA replication, and phosphorylation of E1 appears to regulate protein function and DNA replication. Serine 584 of bovine papillomavirus E1 is in a conserved motif resembling a CK2 consensus site, and is phosphorylated by CK2 in vitro. Mutation of serine 584 to alanine eliminates replication of the viral genome in transient replication assays. Wild-type and mutant E1 proteins were expressed from recombinant baculoviruses and used to assess biochemical functions of the amino acid 584 substitution. Helicase enzyme activity, E1 binding to the viral E2 protein and to cellular DNA polymerase alpha-primase were all unaffected in the mutant protein. Binding of E1 to viral replication origin DNA sequences was reduced in the mutant, but not eliminated. The carboxyl-terminal region of the protein appears to play a role in regulating E1 function, and adds to a complex picture emerging for papillomavirus DNA replication control.     

Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins.

Infection with adenovirus mutants carrying either point mutations or deletions in the coding region for the 19-kDa E1B gene product (19K protein) causes degradation of host cell and viral DNAs (deg phenotype) and enhanced cytopathic effect (cyt phenotype). Therefore, one function of the E1B 19K protein is to protect nuclear DNA integrity and preserve cytoplasmic architecture during productive adenovirus infection. When placed in the background of a virus incapable of expressing a functional E1A gene product, however, E1B 19K gene mutations do not result in the appearance of the cyt and deg phenotypes. This demonstrated that expression of the E1A proteins was responsible for inducing the appearance of the cyt and deg phenotypes. By constructing a panel of viruses possessing E1A mutations spanning each of the three E1A conserved regions in conjunction with E1B 19K gene mutations, we mapped the induction of the cyt and deg phenotypes to the amino-terminal region of E1A. Viruses that fail to express conserved region 3 (amino acids 140 to 185) and/or 2, (amino acids 121 to 185) or nonconserved sequences between conserved regions 2 and 1 of E1A (amino acids 86 to 120) were still capable of inducing cyt and deg. This indicated that activities associated with these regions, such as transactivation and binding to the product of the retinoblastoma susceptibility gene, were dispensable for induction of E1A-dependent cytotoxic effects. In contrast, deletion of sequences in the amino terminus of E1A (amino acids 22 to 107) resulted in extragenic suppression of the cyt and deg phenotypes. Therefore, a function affected by deletion of amino acids 22 to 86 of E1A is responsible for exerting cytotoxic effects in virally infected cells. Furthermore, transient high-level expression of the E1A region using a cytomegalovirus promoter plasmid expression vector was sufficient to induce the cyt and deg phenotypes, demonstrating that E1A expression alone is sufficient to exert these cytotoxic effects and that other viral gene products are not involved. Finally, placing E1A expression under the control of a strong promoter did not alter the requirement for E1B in the transformation of primary cells. One possibility is that the E1B 19K protein is required to overcome the cytotoxic effects of E1A protein expression and thereby enable primary cells to become transformed.     

Mapping and characterization of the interaction domains of human papillomavirus type 16 E1 and E2 proteins.

The papillomavirus E1 and E2 proteins are both necessary and sufficient in vivo for efficient origin-dependent viral DNA replication. The ability of E1 and E2 to complex with each other appears to be essential for efficient viral DNA replication. In this study, we used the yeast two-hybrid system and in vitro binding assays to map the domains of the human papillomavirus type 16 (HPV16) E1 and E2 proteins required for complex formation. The amino-terminal 190-amino-acid domain of HPV16 E2 was both required and sufficient for E1 binding. The carboxyl-terminal 229 amino acids of E 1 were essential for binding E2, and the amino-terminal 143 amino acids of HPV16 E1 were dispensable. Although the ability of the E1 minimal domain (amino acids [aa] 421 to 649) to interact with E2 was strong at 4 degrees C, it was significantly reduced at temperatures above 25 degrees C. A larger domain of E1 from aa 144 to 649 bound E2 efficiently at any temperature, suggesting that aa 144 to 420 of E1 may play a role in the HPV16 E1-E2 interaction at physiological temperatures.     

Mutational analysis of the 18-base-pair inverted repeat element at the bovine papillomavirus origin of replication: identification of critical sequences for E1 binding and in vivo replication.

Replication of bovine papillomavirus requires two viral proteins, E1 and E2-TA. Previously we demonstrated that sequences within an imperfect 18-bp inverted repeat (IR) element were sufficient to confer specific binding of the E1 protein to the origin region (S. E. Holt, G. Schuller, and V. G. Wilson, J. Virol. 68:1094-1102, 1994). To identify critical nucleotides for E1 binding and origin function, a series of individual point mutations was constructed at each nucleotide position in the 18-bp IR. Binding of E1 to these point mutations established that both the position of the mutation and the specific nucleotide change were important for the E1-DNA interaction. Equivalent mutations from each half of the IR exhibited similar binding, suggesting that the halves were functionally symmetric for E1 interactions. Each of these mutations was evaluated also for origin function in vivo by a transient-replication assay. No single point mutation eliminated replication capacity completely, though many mutants were severely impaired, demonstrating an important functional contribution for the E1 binding site. Furthermore, E1 binding was not sufficient for replication, as several origin mutants bound E1 well in vitro but replicated poorly in vivo. This suggests that certain nucleotides within the 18-bp IR may be involved in postbinding events necessary for replication initiation. The results with the point mutations suggest that E1-E1 interactions are important for stable complex formation and also indicate that there is some flexibility with regard to formation of a functional E1 replication complex at the origin.     

Chaperone proteins abrogate inhibition of the human papillomavirus (HPV) E1 replicative helicase by the HPV E2 protein

Human papillomavirus (HPV) DNA replication requires the viral origin recognition protein E2 and the presumptive viral replicative helicase E1. We now report for the first time efficient DNA unwinding by a purified HPV E1 protein. Unwinding depends on a supercoiled DNA substrate, topoisomerase I, single-stranded-DNA-binding protein, and ATP, but not an origin. Electron microscopy revealed completely unwound molecules. Intermediates contained two single-stranded loops emanating from a single protein complex, suggesting a bidirectional E1 helicase which translocated the flanking DNA in an inward direction. We showed that E2 protein partially inhibited DNA unwinding and that Hsp70 or Hsp40, which we reported previously to stimulate HPV-11 E1 binding to the origin and promote dihexameric E1 formation, apparently displaced E2 and abolished inhibition. Neither E2 nor chaperone proteins were detected in unwinding complexes. These results suggest that chaperones play important roles in the assembly and activation of a replicative helicase in higher eukaryotes. An E1 mutation in the ATP binding site caused deficient binding and unwinding of origin DNA, indicating the importance of ATP binding in efficient helicase assembly on the origin.     

Cyclin/CDK regulates the nucleocytoplasmic localization of the human papillomavirus E1 DNA helicase

Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.     

E1 recognition sequences in the bovine papillomavirus type 1 origin of DNA replication: interaction between half sites of the inverted repeats.

The E1 protein encoded by bovine papillomavirus type 1 (BPV-1) is required for viral DNA replication, and it binds site specifically to an A/T-rich palindromic sequence within the viral origin of replication. The protein is targeted to this site through cooperative interactions and binding with the virus-encoded E2 protein. To explore the nature of the E1 binding site, we inserted a series of homologous DNA linkers at the center of dyad symmetry within the E1 recognition palindrome. The effects of these modifications indicated that the E1 recognition palindrome can be separated into functional half sites. The series of insertions manifest a phasing relationship with respect to the wild-type BPV-1 genome in that greater biological activity was measured when full integral turns of the DNA helix separated the palindrome than when the separations were half-turns. This phasing pattern of activity was observed to occur in a variety of biological phenotypes, including transformation efficiency, stable plasmid copy number in cell lines established from pooled foci, and transient replication of full-length viral genomes. For replication reporter constructs where E1 and E2 are supplied in trans by the respective expression vectors, distance between the half sites seems to play a major role, yet the phasing relationships are measurable. DNase I protection studies showed that E1 bound very poorly to the construct containing a 5-bp linker, and binding was close to the wild-type level for the 10-bp insertion, consistent with a requirement for a phasing function between half sites with a modulus of 10 bp. Binding to the 15- and 20-bp insertion mutants was weak, but only for the 20-bp insertions was protection over both halves of the palindrome measurable. As it had been previously reported that the 18-bp palindrome contains sufficient nucleotide sequence information for E1 binding, we speculate that a minimal E1 recognition motif is presented in each half site. A comparison between this sequence and that of an upstream region that also binds E1 (the E2RE1 region) revealed a common pentanucleotide motif of APyAAPy. Mutants with substitutions of the ATAAT elements within E2RE1 failed to bind E1 protein. We present models for how repeats of the pentanucleotide sequence may coordinate E1 binding at the dyad symmetry axis of the origin and compare the DNA sequence organization of BPV-1 with those of the simian virus 40 and polyomaviruses at their origins of DNA replication.     

Cellular topoisomerase I modulates origin binding by bovine papillomavirus type 1 E1

In addition to viral proteins E1 and E2, bovine papillomavirus type 1 (BPV1) depends heavily on host replication machinery for genome duplication. It was previously shown that E1 binds to and recruits cellular replication proteins to the BPV1 origin of replication, including DNA polymerase alpha-primase, replication protein A (RPA), and more recently, human topoisomerase I (Topo I). Here, we show that Topo I specifically stimulates the origin binding of E1 severalfold but has no effect on nonorigin DNA binding. This is highly specific, as binding to nonorigin DNA is not stimulated, and other cellular proteins that bind E1, such as RPA and polymerase alpha-primase, show no such effect. The stimulation of E1's origin binding by Topo I is not synergistic with the stimulation by E2. Although the enhanced origin binding of E1 by Topo I requires ATP and Mg2+ for optimal efficiency, ATP hydrolysis is not required. Using an enzyme-linked immunosorbent assay, we showed that the interaction between E1 and Topo I is decreased in the presence of DNA. Our results suggest that Topo I participates in the initiation of papillomavirus DNA replication by enhancing E1 binding to the BPV1 origin.     

Identification of the origin of replication of bovine papillomavirus and characterization of the viral origin recognition factor E1.

Expression of the viral polypeptides E1 and E2 is necessary and sufficient for replication of BPV in mouse C127 cells. By providing these factors from heterologous expression vectors we have identified a minimal origin fragment from BPV that contains all the sequences required in cis for replication of BPV in short term replication assays. This same sequence is also required for stable replication in the context of the entire viral genome. The identified region is highly conserved between different papillomaviruses, and is unrelated to the previously identified plasmid maintenance sequences. The minimal ori sequence contains a binding site for the viral polypeptide E1, which we identify as a sequence specific DNA binding protein, but surprisingly, an intact binding site for the viral transactivator E2 at the ori is not required. The isolated origin shows an extended host region for replication and replicates efficiently in both rodent and primate cell lines.     

Identification of domains of the human papillomavirus type 11 E1 helicase involved in oligomerization and binding to the viral origin

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 single-stranded 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.