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.     

Dynamic localization of the human papillomavirus type 11 origin binding protein E2 through mitosis while in association with the spindle apparatus

Papillomaviral DNA replicates as extrachromosomal plasmids in squamous epithelium. Viral DNA must segregate equitably into daughter cells to persist in dividing basal/parabasal cells. We have previously reported that the viral origin binding protein E2 of human papillomavirus types 11 (HPV-11), 16, and 18 colocalized with the mitotic spindles. In this study, we show the localization of the HPV-11 E2 protein to be dynamic. It colocalized with the mitotic spindles during prophase and metaphase. At anaphase, it began to migrate to the central spindle microtubules, where it remained through telophase and cytokinesis. It was additionally observed in the midbody at cytokinesis. A peptide spanning residues 285 to 308 in the carboxyl-terminal domain of HPV-11 E2 (E2C) is necessary and sufficient to confer localization on the mitotic spindles. This region is conserved in HPV-11, -16, and -18 and bovine papillomavirus type 4 (BPV-4) E2 and is also required for the respective E2C to colocalize with the mitotic spindles. The E2 protein of bovine papillomavirus type 1 is tethered to the mitotic chromosomes via the cellular protein Brd4. However, the HPV-11 E2 protein did not associate with Brd4 during mitosis. Lastly, a chimeric BPV-1 E2C containing the spindle localization domain from HPV-11 E2C gained the ability to localize to the mitotic spindles, whereas the reciprocal chimera lost the ability. We conclude that this region of HPV E2C is critical for localization with the mitotic apparatus, enabling the HPV DNA to sustain persistent infections.     

The DNA-binding domain of human papillomavirus type 18 E1. Crystal structure, dimerization, and DNA binding

High risk types of human papillomavirus, such as type 18 (HPV-18), cause cervical carcinoma, one of the most frequent causes of cancer death in women worldwide. DNA replication is one of the central processes in viral maintenance, and the machinery involved is an excellent target for the design of antiviral therapy. The papillomaviral DNA replication initiation protein E1 has origin recognition and ATP-dependent DNA melting and helicase activities, and it consists of a DNA-binding domain and an ATPase/helicase domain. While monomeric in solution, E1 binds DNA as a dimer. Dimerization occurs via an interaction of hydrophobic residues on a single alpha-helix of each monomer. Here we present the crystal structure of the monomeric HPV-18 E1 DNA-binding domain refined to 1.8-A resolution. The structure reveals that the dimerization helix is significantly different from that of bovine papillomavirus type 1 (BPV-1). However, we demonstrate that the analogous residues required for E1 dimerization in BPV-1 and the low risk HPV-11 are also required for HPV-18 E1. We also present evidence that the HPV-18 E1 DNA-binding domain does not share the same nucleotide and amino acid requirements for specific DNA recognition as BPV-1 and HPV-11 E1.     

The human papillomavirus type 6 and 16 E5 proteins are membrane-associated proteins which associate with the 16-kilodalton pore-forming protein.

The human papillomavirus (HPV) E5 proteins are predicted from DNA sequence analysis to be small hydrophobic molecules, and the HPV type 6 (HPV-6) and HPV-11 E5 proteins share several structural similarities with the bovine papillomavirus type 1 (BPV-1) E5 protein. Also similar to the BPV-1 E5 protein, the HPV-6 and HPV-16 E5 proteins exhibit transforming activity when assayed on NIH 3T3 and C127 cells. In this study, we expressed epitope-tagged E5 proteins from both the "low-risk" HPV-6 and the "high-risk" HPV-16 in order to permit their immunologic identification and biochemical characterization. While the HPV-6 and HPV-16 E5 proteins fail to form disulfide-linked dimers and oligomers, they did resemble the BPV-1 E5 protein in their intracellular localization to the Golgi apparatus, endoplasmic reticulum, and nuclear membranes. In addition, the HPV E5 proteins also bound to the 16-kDa pore-forming protein component of the vacuolar ATPase, a known characteristic of the BPV-1 E5 protein. These studies reveal a common intramembrane localization and potential cellular protein target for both the BPV and HPV E5 proteins.     

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.     

Characterization of recombinant HPV6 and 11 E1 helicases: effect of ATP on the interaction of E1 with E2 and mapping of a minimal helicase domain

To better characterize the enzymatic activities required for human papillomavirus (HPV) DNA replication, the E1 helicases of HPV types 6 and 11 were produced using a baculovirus expression system. The purified wild type proteins and a version of HPV11 E1 lacking the N-terminal 71 amino acids, which was better expressed, were found to be hexameric over a wide range of concentrations and to have helicase and ATPase activities with relatively low values for K(m)(ATP) of 12 microm for HPV6 E1 and 6 microm for HPV11 E1. Interestingly, the value of K(m)(ATP) was increased 7-fold in the presence of the E2 transactivation domain. In turn, ATP was found to perturb the co-operative binding of E1 and E2 to DNA. Mutant and truncated versions of in vitro translated E1 were used to identify a minimal ATPase domain composed of the C-terminal 297 amino acids. This fragment was expressed, purified, and found to be fully active in ATP hydrolysis, single-stranded DNA binding, and unwinding assays, despite lacking the minimal origin-binding domain.     

Transient replication of human papillomavirus DNAs.

Information on papillomavirus DNA replication has primarily derived from studies with bovine papillomavirus type 1 (BPV-1). Our knowledge of DNA replication of the human papillomaviruses (HPVs) is quite limited, in part because of the lack of a cell culture system capable of supporting the stable replication of HPV DNA. This study demonstrates that the full-length genomic DNAs of HPV types 11 and 18 (HPV-11 and HPV-18), but not HPV-16, are able to replicate transiently after transfection into several different human squamous cell carcinoma cell lines. This system was used to identify the viral cis and trans elements required for DNA replication. The viral origins of replication were localized to a region of the viral long control region. Like BPV-1, E1 and E2 were the only viral factors required in trans for the replication of plasmids containing the origin. Cotransfection of a plasmid expressing the E1 open reading frame (ORF) from HPV-11 with a plasmid that expresses the E2 ORF from HPV-6, HPV-11, HPV-16, or HPV-18 supported the replication of plasmid DNAs containing the origin regions of HPV-11, HPV-16, or HPV-18, indicating that there are functions shared among the corresponding E1 and E2 proteins and origins of these viruses. Although HPV-16 genomic DNA did not replicate by itself under experimental conditions that supported the replication of HPV-11 and HPV-18 genomic DNAs, expression of the HPV-16 early region functions from a strong heterologous promoter supported the replication of a cotransfected plasmid containing the HPV-16 origin of replication. This finding suggests that the inability of the HPV-16 genomic DNA to replicate transiently in the cell lines tested was most likely due to insufficient expression of the viral E1 and/or E2 genes required for DNA replication.     

The E2 binding sites determine the efficiency of replication for the origin of human papillomavirus type 18.

Human papillomaviruses (HPV-s) have been shown to possess transforming and immortalizing activity for many different, mainly keratinocyte cell lines and they have been detected in 90% of anogenital cancer tissues, which suggests a causative role in the induction of anogenital and other tumours. We have exploited a quantitative assay to identify and characterize the origin of replication of the human papillomavirus type 18 (HPV-18), one of the most prevalent types in the high-risk HPV group. Replication of HPV origin fragments was studied transiently by cotransfection with a protein expression vector providing replication proteins E1 and E2. We have localized the HPV-18 origin to nucleotides 7767-119. This region contains three E2 binding sites and an essential A/T rich DNA region (nucleotides 9-35) that is partly homologous to the E1 binding site found in bovine papillomavirus type 1 (BPV-1) genome. At least one of the three E2 binding sites was absolutely required for origin function; addition of other E2 sites had cooperative stimulating effect. This is the first quantitative analysis of the E2 binding sites for papillomavirus replication.     

A C-Terminal Helicase Domain of the Human Papillomavirus E1 Protein Binds E2 and the DNA Polymerase α-Primase p68 Subunit

The human papillomavirus (HPV) E1 and E2 proteins bind cooperatively to the viral origin of replication (ori), forming an E1-E2-ori complex that is essential for initiation of DNA replication. All other replication proteins, including DNA polymerase α-primase (polα-primase), are derived from the host cell. We have carried out a detailed analysis of the interactions of HPV type 16 (HPV-16) E1 with E2, ori, and the four polα-primase subunits. Deletion analysis showed that a C-terminal region of E1 (amino acids [aa] 432 to 583 or 617) is required for E2 binding. HPV-16 E1 was unable to bind the ori in the absence of E2, but the same C-terminal domain of E1 was sufficient to tether E1 to the ori via E2. Of the polα-primase subunits, only p68 bound E1, and binding was competitive with E2. The E1 region required (aa 397 to 583) was the same as that required for E2 binding but additionally contained 34 N-terminal residues. In confirmation of these differences, we found that a monoclonal antibody, mapping adjacent to the N-terminal junction of the p68-binding region, blocked E1-p68 but not E1-E2 binding. Sequence alignments and secondary-structure prediction for HPV-16 E1 and other superfamily 3 (SF3) viral helicases closely parallel the mapping data in suggesting that aa 439 to 623 constitute a discrete helicase domain. Assuming a common nucleoside triphosphate-binding fold, we have generated a structural model of this domain based on the X-ray structures of the hepatitis C virus and Bacillus stearothermophilus (SF2) helicases. The modelling closely matches the deletion analysis in suggesting that this region of E1 is indeed a structural domain, and our results suggest that it is multifunctional and critical to several stages of HPV DNA replication.     

Interaction of the bovine papillomavirus E2 protein with Brd4 tethers the viral DNA to host mitotic chromosomes

The papillomavirus E2 protein tethers viral genomes to host mitotic chromosomes to ensure genome maintenance. We have identified the bromodomain protein Brd4 as a major cellular interacting partner of the bovine papillomavirus E2. Brd4 associates with mitotic chromosomes and colocalizes with E2 on mitotic chromosomes. The site of E2 binding maps to the C-terminal domain of Brd4. Expression of this C-terminal Brd4 domain functions in a dominant-negative manner to abrogate the colocalization of E2 with Brd4 on mitotic chromosomes, to block association of the viral episomes with Brd4, and to inhibit BPV-1 DNA-mediated cellular transformation. Brd4 also associates with HPV16 E2, indicating that Brd4 binding may be a shared property of all papillomavirus E2 proteins. The interaction of E2 with Brd4 is required to ensure the tethering of viral genomes to the host mitotic chromosomes for persistence of viral episomes in PV-infected cells.     

Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product.

The E7 proteins encoded by the human papillomaviruses (HPVs) associated with anogenital lesions share significant amino acid sequence homology. The E7 proteins of these different HPVs were assessed for their ability to form complexes with the retinoblastoma tumor suppressor gene product (p105-RB). Similar to the E7 protein of HPV-16, the E7 proteins of HPV-18, HBV-6b and HPV-11 were found to associate with p105-RB in vitro. The E7 proteins of HPV types associated with a high risk of malignant progression (HPV-16 and HPV-18) formed complexes with p105-RB with equal affinities. The E7 proteins encoded by HPV types 6b and 11, which are associated with clinical lesions with a lower risk for progression, bound to p105-RB with lower affinities. The E7 protein of the bovine papillomavirus type 1 (BPV-1), which does not share structural similarity in the amino terminal region with the HPV E7 proteins, was unable to form a detectable complex with p105-RB. The amino acid sequences of the HPV-16 E7 protein involved in complex formation with p105-RB in vitro have been mapped. Only a portion of the sequences that are conserved between the HPV E7 proteins and AdE1A were necessary for association with p105-RB. Furthermore, the HPV-16 E7-p105-RB complex was detected in an HPV-16-transformed human keratinocyte cell line.     

Inhibition of human papillomavirus DNA replication by small molecule antagonists of the E1-E2 protein interaction

Human papillomavirus (HPV) DNA replication is initiated by recruitment of the E1 helicase by the E2 protein to the viral origin. Screening of our corporate compound collection with an assay measuring the cooperative binding of E1 and E2 to the origin identified a class of small molecule inhibitors of the protein interaction between E1 and E2. Isothermal titration calorimetry and changes in protein fluorescence showed that the inhibitors bind to the transactivation domain of E2, the region that interacts with E1. These compounds inhibit E2 of the low risk HPV types 6 and 11 but not those of high risk HPV types or of cottontail rabbit papillomavirus. Functional evidence that the transactivation domain is the target of inhibition was obtained by swapping this domain between a sensitive (HPV11) and a resistant (cottontail rabbit papillomavirus) E2 type and by identifying an amino acid substitution, E100A, that increases inhibition by approximately 10-fold. This class of inhibitors was found to antagonize specifically the E1-E2 interaction in vivo and to inhibit HPV DNA replication in transiently transfected cells. These results highlight the potential of the E1-E2 interaction as a small molecule antiviral target.