To replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination. IMPORTANCEHuman papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the alleviation of disease burden on the current, and future, generations. Targeting viral DNA replication that is mediated by two viral proteins, E1 and E2, in association with cellular proteins such as TopBP1 and Brd4 would have therapeutic benefits. This report suggests a role for these cellular proteins in the initiation of viral DNA replication by HPV16 E1-E2 but not for continuing replication. This is important if viral replication is to be effectively targeted; we need to understand the viral and cellular proteins required at each phase of viral DNA replication so that it can be effectively disrupted. Human papillomaviruses (HPVs) are double-stranded DNA viruses that infect the epithelium and cause a variety of human diseases. Human papillomavirus 16 (HPV16) is the most commonly found HPV in cervical cancer (found in around 50% of cases) and also in head and neck cancer (around 90% of the HPVpositive cases) (see reference 1 for a recent review). Two viral proteins, E1 and E2, are required for viral replication. E2 has a carboxyl terminus DNA binding and d...

HPV-16 is the major causes of cervical cancer. Persistence of infection is a necessary event for progression of the infection to cancer. Among other factors, virus persistence is due the viral proteins fighting the immune response. HPV-16 E5 down-regulates MHC/HLA class I, which is much reduced on the cell surface and accumulates in the Golgi apparatus in cells expressing E5. This effect is observed also in W12 cells, which mimic early cervical intraepithelial progression to cervical cancer. The functional effect of MHC I down-regulation on human CD8 T cells is not known, because of the need for HLA-matched, HPV-specific T cells that recognise E5 expressing-cells. Here we employ a heterologous cell/MHC I system which uses mouse cells expressing both E5 and HLA-A2, and A2-restricted CTLs; we show that the E5-induced reduction of HLA-A2 has a functional impact by reducing recognition of E5 expressing cells by HPV specific CD8+ T cells.

cleoside analogues, [8][9][10] the only established treatment option Hepatitis B virus (HBV) replicates via an intermediate to prevent reinfection is the administration of polyclonal hep-RNA step. High frequency of polymerase errors with adatitis B surface antigen antibody (anti-HBs) (hepatitis B imditional selection pressure leads to mutations in the mune globulin [HBIG]). HBIG reduces the rate of reinfection HBV genome. We investigated the number, type, and anfrom about 90% to less than 30% and improves the long-term tigenic effects of mutations in the coding region of the outcome of patients who underwent OLT for HBV-related HBV surface antigen in eight patients who underwent disease. 2-4orthotopic liver transplantation (OLT) for HBV-related A number of explanations for graft infection have been end-stage liver disease and were experiencing infection proposed. First, virions from the explanted liver are circulatof the graft and who received hepatitis B surface antigen ing in the blood during or soon after transplantation. Second, antibody (anti-HBs) prophylaxis (hepatitis B immune virions are replicating at extrahepatic sites. HBIG interferes globulin [HBIG]) after OLT. Controls were chronic HBV with this process and probably prevents virions from entering patients who underwent kidney transplantation and rehepatocytes. 11 The mechanisms that lead to reinfection in ceived the same immunosuppressive regime but no 30% of patients receiving HBIG after OLT are not under-HBIG. The S-gene was amplified from serum before and stood. after transplantation, sequenced, and changes in the ge-HBV employs reverse transcriptase for its replication; this nome were analyzed. In the five patients who experilacks proofreading capability, and thus leads to a higher enced reinfection while receiving anti-HBs, clear mutanumber of mutations in the HBV genome. 12 Some of these tions occurred in the S-gene. In the patient who did not variants may have a replication advantage and become domireceive HBIG and those who experienced reinfection nant. From the pool of variants with similar replication poonly after termination of HBIG, no mutations were tential, some will be positively selected by forces such as found in the S-gene. In the kidney recipients, mutations the humoral or cellular immune response 13; these are termed in the S-gene occurred in only one of eight patients. Beescape mutants. Evolution of viruses under antibody prescause the a determinant contains neutralizing epitopes, sure, either monoclonal or polyclonal, has been well studied this region was chosen for antibody binding to quantify both in vitro and in vivo. The addition of neutralizing antibodantigenic effects of the mutations. The two patients who ies to virus cultures regularly results in isolates that are not selected mutations in the a determinant and became reneutralized by the added antibodies. This is particularly true infected while receiving HBIG had reduced antibody with monoclonal reagents, because the change in viral antibinding after OLT. Our results...

bIn human papillomavirus DNA replication, the viral protein E2 forms homodimers and binds to 12-bp palindromic DNA sequences surrounding the origin of DNA replication. Via a protein-protein interaction, it then recruits the viral helicase E1 to an A/T-rich origin of replication, whereupon a dihexamer forms, resulting in DNA replication initiation. In order to carry out DNA replication, the viral proteins must interact with host factors that are currently not all known. An attractive cellular candidate for regulating viral replication is TopBP1, a known interactor of the E2 protein. In mammalian DNA replication, TopBP1 loads DNA polymerases onto the replicative helicase after the G 1 -to-S transition, and this process is tightly cell cycle controlled. The direct interaction between E2 and TopBP1 would allow E2 to bypass this cell cycle control, resulting in DNA replication more than once per cell cycle, which is a requirement for the viral life cycle. We report here the generation of an HPV16 E2 mutant compromised in TopBP1 interaction in vivo and demonstrate that this mutant retains transcriptional activation and repression functions but has suboptimal DNA replication potential. Introduction of this mutant into a viral life cycle model results in the failure to establish viral episomes. The results present a potential new antiviral target, the E2-TopBP1 interaction, and increase our understanding of the viral life cycle, suggesting that the E2-TopBP1 interaction is essential. There are more than 100 types of human papillomavirus (HPV) involved in a host of epithelial lesions, ranging from hand warts and genital warts to cervical cancer (69). So-called high-risk HPVs are those associated with cancer, and type 16 is the most commonly detected, being present in ca. 50% of cervical carcinomas and increasingly detected in head and neck cancers (30). All HPV encode two proteins, E1 and E2, required for replication of their double-stranded DNA genome in association with cellular partner proteins. The E2 protein forms homodimers and binds to 12-bp palindromic sequences surrounding the origin of replication and via a protein-protein interaction recruits the E1 protein to the A/T-rich origin (9,40,61). E1 then forms a dihexameric helicase that interacts with the cellular DNA polymerase machinery, resulting in DNA replication initiation (36,38,46,55). The origin of replication is located in the long control region (LCR), a noncoding part of the genome that controls the initial transcription from the viral genome by cellular factors (50). The E2 protein can also regulate viral genome transcription; it can act as either an activator or a repressor of viral oncogene expression depending upon E2 levels and the cell type under study (10,15,60). The carboxyl terminus domain of E2 is required for homodimerization and DNA binding, while the amino terminus interacts with E1 and a number of cellular transcription factors (16,47,54,56,63). E2 can also associate with mitotic chromatin and is proposed as a viral genome segregation fact...

The distribution and temporal and clinical features of amino acid substitutions of the core protein of hepatitis B (HB) virus were analyzed, using at least 2 sequential samples from 27 patients. Six patients seroconverted from HBe antigen (HBeAg)-positive to anti-HBe-positive (3 went into remission), and 21 were continuously anti-HBe positive with progressive hepatitis. Precore mutations, which terminate HBeAg translation, all appeared by the second sample. Most core mutations occurred between the first and second samples; significantly fewer occurred after the second. In seroconverters who went into remission, mutations occurred in the T helper epitope from aa 50 to 69 (P = .00045); for anti-HBe-positive patients with ongoing disease, mutations occurred in B cell epitopes (P = .0007 for aa 74-83). An ineffective anti-HBc B cell response accounts for ongoing disease and selection of mutations after seroconversion. In those who remit, mutations in the major T helper epitope allow immune escape, thus minimizing immune-mediated hepatitis.

Integration of human papillomaviruses into that of the host promotes genomic instability and progression to cancer; factors that promote integration remain to be fully identified. DNA damage agents can promote double strand breaks during DNA replication providing substrates for integration and we investigated the ability of DNA damage to regulate HPV E1 and E2 mediated DNA replication. Results demonstrate that HPV E1 and E2 replication is not arrested following DNA damage, both in vivo and in vitro, while replication by SV40 Large T antigen is arrested and ATR is the candidate kinase for mediating the arrest. LTAg is a target for PIKK DNA damage signalling kinases, while E1 is not. We propose that the failure of E1 to be targeted by PIKKs allows HPV replication in the presence of DNA damaging agents. Such replication will result in double strand breaks in the viral genome ultimately promoting viral integration and cervical cancer.

Human papillomavirus 16 (HPV16) is causative in human cancer. The E2 protein regulates transcription from and replication of the viral genome; the role of E2 in regulating the host genome has been less well studied. We have expressed HPV16 E2 (E2) stably in U2OS cells; these cells tolerate E2 expression well and gene expression analysis identified 74 genes showing differential expression specific to E2. Analysis of published gene expression data sets during cervical cancer progression identified 20 of the genes as being altered in a similar direction as the E2 specific genes. In addition, E2 altered the splicing of many genes implicated in cancer and cell motility. The E2 expressing cells showed no alteration in cell growth but were altered in cell motility, consistent with the E2 induced altered splicing predicted to affect this cellular function. The results present a model system for investigating E2 regulation of the host genome.

For the first time this report demonstrates aberrant expression of the TopBP1 protein in breast carcinoma. We propose TOPBP1 as a breast cancer susceptibility gene.