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

In vitro DNA binding results from a series of E1 proteins containing amino-terminal or carboxy-terminal truncations indicated that sequences between amino acids 121 and 284 were critical for origin binding. Additional binding experiments with E1 proteins containing internal, in-frame insertions or deletions confirmed the importance of the region defined by truncated E1 proteins and also demonstrated that downstream sequences were not required for binding activity in the context of the full-length E1 protein. On the basis of mapping results from the E1 mutants, a clone (pE1(121-311)) was constructed that expressed E1 amino acids within the approximate boundaries of the critical sequences for DNA binding. The E1(121-311) protein retained origin-specific DNA binding, confirming that this region was not only necessary but was also sufficient for origin recognition. In addition to origin binding, E1(121-311) bound E2 protein in a cold-sensitive manner. Therefore, DNA binding and E2 binding activities colocalize to a 191-amino-acid functional domain derived from the amino-terminal half of the E1 protein. Finally, three E1 proteins with mutations in this region all lacked DNA binding activity and were all defective for in vivo replication. Two of these E1 mutants retained E2 binding capability, demonstrating that origin recognition by E1 is critical for replication and cannot necessarily be rescued by an interaction with E2 protein.     

Human papillomavirus DNA replication. Interactions between the viral E1 protein and two subunits of human dna polymerase alpha/primase

Papovaviruses are valuable models for the study of DNA replication in higher eukaryotic organisms, as they depend on host factors for replication of their DNA. In this study we investigate the interactions between the human papillomavirus type 11 (HPV-11) origin recognition and initiator protein E1 and human polymerase alpha/primase (pol alpha/primase) subunits. By using a variety of physical assays, we show that both 180- (p180) and 70-kDa (p70) subunits of pol alpha/primase interact with HPV-11 E1. The interactions of E1 with p180 and p70 are functionally different in cell-free replication of an HPV-11 origin-containing plasmid. Exogenously added p180 inhibits both E2-dependent and E2-independent cell-free replication of HPV-11, whereas p70 inhibits E2-dependent but stimulates E2-independent replication. Our experiments indicate that p70 does not physically interact with E2 and suggest that it may compete with E2 for binding to E1. A model of how E2 and p70 sequentially interact with E1 during initiation of viral DNA replication is proposed.     

Hepatitis C virus core protein interacts with the cytoplasmic tail of lymphotoxin-beta receptor

Hepatitis C virus (HCV) core protein is a multifunctional protein. We examined whether it can interact with cellular proteins, thus contributing to viral pathogenesis. Using the HCV core protein as a bait to screen a human liver cDNA library in a yeast two-hybrid screening system, we have isolated several positive clones encoding cellular proteins that interact with the HCV core protein. Interestingly, more than half of these clones encode the cytoplasmic domain of lymphotoxin-beta receptor (LT betaR), which is a member of the tumor necrosis factor receptor family. Their binding was confirmed by in vitro glutathione S-transferase fusion protein binding assay and protein-protein blotting assay to be direct and specific. The binding sites were mapped within a 58-amino-acid region of the cytoplasmic tail of LT betaR. The binding site in the HCV core protein was localized within amino acid residues 36 to 91 from the N terminus, corresponding to the hydrophilic region of the protein. In mammalian cells, the core protein was found to be associated with the membrane-bound LT betaR. Since the LT betaR is involved in germinal center formation and developmental regulation of peripheral lymphoid organs, lymph node development, and apoptotic signaling, the binding of HCV core protein to LT betaR suggests the possibility that this viral protein has an immunomodulating function and may explain the mechanism of viral persistence and pathogenesis of HCV.     

Competition for DNA binding sites between the short and long forms of E2 dimers underlies repression in bovine papillomavirus type 1 DNA replication control

Papillomaviruses establish a long-term latency in vivo by maintaining their genomes as nuclear plasmids in proliferating cells. Bovine papillomavirus type 1 encodes two proteins required for viral DNA replication: the helicase E1 and the positive regulator E2. The homodimeric E2 is known to cooperatively bind to DNA with E1 to form a preinitiation complex at the origin of DNA replication. The virus also codes for two short forms of E2 that can repress viral functions when overexpressed, and at least one copy of the repressor is required for stable plasmid maintenance in transformed cells. Employing a tetracycline-regulated system to control E1 and E2 production from integrated loci, we show that the short form of E2 negatively regulates DNA replication. We also found that the short form could repress replication in a cell-free replication system and that the repression requires the DNA binding domain of the protein. In contrast, heterodimers of the short and long forms were activators and, by footprint analysis, were shown to be as potent as homodimeric E2 in loading E1 to its cognate site. DNA binding studies show that when E1 levels are low and are dependent upon E2 for occupancy of the origin site, the repressor can block E1-DNA interactions. We conclude that DNA replication modulation results from competition between the different forms of E2 for DNA binding. Given that heterodimers are active and that the repressor form of E2 shows little cooperativity with E1 for DNA binding, this protein is a weak repressor.     

DNA replication of human papillomavirus type 31 is modulated by elements of the upstream regulatory region that lie 5' of the minimal origin

The viral replication factors E1 and E2 of papillomaviruses are necessary and sufficient to replicate plasmids containing the minimal origin of DNA replication in transient assays. Under physiological conditions, the upstream regulatory region (URR) governs expression of the early viral genes. To determine the effect of URR elements on E1 and E2 expression specifically, and on the regulation of DNA replication during the various phases of the viral life cycle, we carried out a systematic replication study with entire genomes of human papillomavirus type 31 (HPV31), a high-risk oncogenic type. We constructed a series of URR deletions, spacer replacements, and point mutations to analyze the role of the keratinocyte enhancer (KE) element, the auxiliary enhancer (AE) domain, and the L1-proximal end of the URR (5'-URR domain) in DNA replication during establishment, maintenance, and vegetative viral DNA amplification. Using transient and stable replication assays, we demonstrate that the KE and AE are necessary for efficient E1 and E2 gene expression and that the KE can also directly modulate viral replication. KE-mediated activation of replication is dependent on the position and orientation of the element. Mutation of either one of the four Ap1 sites, the single Sp1 site, or the binding site for the uncharacterized footprint factor 1 reduced replication efficiency through decreased expression of E1 and E2. Furthermore, the 5'-URR domain and the Oct1 DNA binding site are dispensable for viral replication, since such HPV31 mutants are able to replicate efficiently in a transient assay, maintain a stable copy number over several cell generations, and amplify viral DNA under vegetative conditions. Interestingly, deletion of the 5'-URR domain leads to increased transient and stable replication levels. These findings suggest that elements in the HPV31 URR outside the minimal origin modulate viral replication through both direct and indirect mechanisms.     

Association of the human papillomavirus type 11 E1 protein with histone H1

The E1 and E2 proteins are the only virus-encoded factors required for human papillomavirus (HPV) DNA replication. The E1 protein is a DNA helicase responsible for initiation of DNA replication at the viral origin. Its recruitment to the origin is facilitated by binding to E2, for which specific recognition elements are located at the origin. The remaining replication functions for the virus, provided by the host cell's replication machinery, may be mediated by further interactions with E1 and E2. Histone H1 was identified as an HPV type 11 (HPV-11) E1-binding protein by far-Western blotting and by microsequence analyses of a 34-kDa protein purified by E1 affinity chromatography. E1 also bound in vitro to H1 isolated under native conditions in association with intact nucleosomes. In addition, E1 and H1 were coimmunoprecipitated by an E1 antiserum from a nuclear extract prepared from cells expressing recombinant E1. Bound H1 was displaced from HPV-11 DNA by the addition of E1, suggesting that E1 can promote replication initiation and elongation by alteration of viral chromatin structure and disruption of nucleosomes at the replication fork. Furthermore, a region of the HPV-11 genome containing the origin of replication was identified which had weaker affinity for H1 than that of the remaining genome. This result suggests that the presence of a DNA structure at or near the HPV origin facilitates initiation of DNA replication by exclusion of H1. These results are similar to those of studies of simian virus 40 DNA replication, in which a large T antigen-H1 interaction and an H1-resistant region at the origin of DNA replication have also been demonstrated.     

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.     

Human papillomavirus DNA replication compartments in a transient DNA replication system

Many DNA viruses replicate their genomes at nuclear foci in infected cells. Using indirect immunofluorescence in combination with fluorescence in situ hybridization, we colocalized the human papillomavirus (HPV) replicating proteins E1 and E2 and the replicating origin-containing plasmid to nuclear foci in transiently transfected cells. The host replication protein A (RP-A) was also colocalized to these foci. These nuclear structures were identified as active sites of viral DNA synthesis by bromodeoxyuridine (BrdU) pulse-labeling. Unexpectedly, the great majority of RP-A and BrdU incorporation was found in these HPV replication domains. Furthermore, E1, E2, and RP-A were also colocalized to nuclear foci in the absence of an origin-containing plasmid. These observations suggest a spatial reorganization of the host DNA replication machinery upon HPV DNA replication or E1 and E2 expression. Alternatively, viral DNA replication might be targeted to host nuclear domains that are active during the late S phase, when such domains are limited in number. In a fraction of cells expressing E1 and E2, the promyelocytic leukemia protein, a component of nuclear domain 10 (ND10), was either partially or completely colocalized with E1 and E2. Since ND10 structures were recently hypothesized to be sites of bovine papillomavirus virion assembly, our observation suggests that HPV DNA amplification might be partially coupled to virion assembly.     

The 23-kilodalton E1 phosphoprotein of bovine papillomavirus type 1 is nonessential for stable plasmid replication in murine C127 cells

The 23-kDa protein encoded by the 5' segment of the E1 open reading frame of bovine papillomavirus type 1 (BPV1) was previously ascribed a negative regulatory function for the replication of viral plasmid DNA. However, results from recent functional and biochemical studies do not readily support this genetic assignment. Therefore, we have reassessed the role of this protein in papillomavirus DNA replication by using a mutant of BPV1 which is unable to express this E1 protein. This mutant viral DNA was found to replicate extrachromosomally with stability and copy number per cell similar to those of wild-type plasmid DNA. Thus, the absence of expression of the 23-kDa E1 protein did not lead to deregulated viral plasmid replication. We conclude that the 23-kDa E1 protein is nonessential for stable plasmid replication.