Edward S. Dornan

HPV-16 E5 down-regulates expression of surface HLA class I and reduces recognition by CD8 T cells

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.

Hepatitis B Virus Core Protein Mutations Are Concentrated in B Cell Epitopes in Progressive Disease and in T Helper Cell Epitopes during Clinical Remission

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.

Aberrant expression of TopBP1 in breast cancer.

Aims:  The TopBP1 protein includes eight BRCT domains (originally identified in BRCA1) and has homology with BRCA1 over the carboxyl terminal half of the protein. The aim of this study was to determine whether TopBP1 is aberrantly expressed in breast cancer.

The E5 protein of BPV-4 interacts with the heavy chain of MHC class I and irreversibly retains the MHC complex in the Golgi apparatus

BPV-4 E5 inhibits transcription of the bovine MHC class I heavy chain (HC) gene, increases degradation of HC and downregulates surface expression of MHC class I by retaining the complex in the Golgi apparatus (GA). Here we report that transcription inhibition can be alleviated by interferon treatment and the degradation of HC can be reversed by treatment with inhibitors of proteasomes and lysosomes. However, the inhibition of transport of MHC class I to the cell surface is irreversible. We show that E5 is capable of physically interacting with HC. Together with the inhibition of the vacuolar ATPase (due to the interaction between E5 and 16k subunit c), the interaction between E5 and HC is likely to be responsible for retention of MHC class I in the GA. C-terminus deletion mutants of E5 are incapable of either downregulating surface MHC class I or interacting with HC, establishing that the C-terminus domain of E5 is important in the inhibition of MHC class I.

Evidence supporting a role for TopBP1 and Brd4 in the initiation but not continuation of human papillomavirus 16 E1/E2 mediated DNA replication

ABSTRACT 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. IMPORTANCE Human 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.

An Interaction between Human Papillomavirus 16 E2 and TopBP1 Is Required for Optimum Viral DNA Replication and Episomal Genome Establishment

ABSTRACT In 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 G1-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.

Human papillomavirus E1 and E2 mediated DNA replication is not arrested by DNA damage signalling

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.

UVB irradiation reduces the half-life and transactivation potential of the human papillomavirus 16 E2 protein

Human papillomaviruses (HPV) are causative agents of human cancers including those of the cervix and also of the head and neck; HPV16 is the most commonly found type in these diseases. The viral E2 protein regulates transcription from the viral genome by interacting with DNA-binding sequences in the HPV transcriptional control region; it also regulates replication by interacting with and recruiting the HPV replication factor E1 to the viral origin. Therefore, E2 is essential for the viral life cycle. The E2 protein interacts with several proteins involved in the cellular response to DNA damage including p53, TopBP1, and PARP. We therefore set out to establish whether DNA-damaging agents can regulate E2 activity. Here we show that UVB irradiation downregulates transcriptional activity of both HPV16 and HPV8 E2, while hydroxyurea and etoposide do not. This downregulation of E2 activity is independent of p53 function as it occurs in p53 wild type and null cell types as well as in the presence of functional HPV16 E6 that degrades p53. Using stable cell lines expressing E2 we show that this downregulation of E2 function by UVB is due to a reduction of the E2 protein half-life. The identification of the pathway(s) through which UVB downregulates E2 transcriptional activity and protein levels will present a novel target for the treatment of HPV-related diseases.

The human papillomavirus 16 E2 protein is stabilised in S phase

The human papillomavirus 16 E2 protein regulates transcription from, and replication of, the viral genome and is also required for segregation of the viral genome via interaction with mitotic bodies. To regulate DNA replication E2 interacts with sequences around the origin of replication and recruits the viral helicase E1 via a protein-protein interaction, which then initiates viral genome replication. The replication role of E2 must originally function in a host cell S phase. In this report, we demonstrate that E2 is stabilised in the S phase of the cell cycle and that this stabilisation is accompanied by an increase in phosphorylation of the protein. This increased phosphorylation and stability are likely required for optimum viral DNA replication and therefore identification of the enzymes involved in regulating these properties of E2 will provide targets for therapeutic intervention in the viral life cycle. Preliminary studies have identified E2 as a Cdk2 substrate demonstrating this enzyme as a candidate kinase for mediating the in vivo phosphorylation of HPV16 E2.

TopBP1 contains a transcriptional activation domain suppressed by two adjacent BRCT domains.

TopBP1 has eight BRCT [BRCA1 (breast-cancer susceptibility gene 1) C-terminus] domains and is involved in initiating DNA replication, and DNA damage checkpoint signalling and repair. Several BRCT-domain-containing proteins involved in mediating DNA repair have transcriptional regulatory domains, and as demonstrated for BRCA1 these regulatory domains are important in mediating the functions of these proteins. These transcriptional regulatory processes involve modification of chromatin, and recent evidence has clearly demonstrated that the ability to modify chromatin plays an important role in regulating DNA damage signalling and repair. Here we report the identification of a TopBP1 transcriptional activation domain that is rich in hydrophobic residues, interspersed with acidic amino acids, characteristics that are typical of transcriptional activation domains identified previously. Two adjacent repressor domains encoded by BRCT2 and BRCT5 silence this activator and experiments suggest that these repressors actively recruit repressor complexes. Both the activator and BRCT2 repressor domains function in yeast. The present study identifies several chromatin modification domains encoded by TopBP1, and the implications of these findings are discussed in the context of the DNA damage response and the understanding of TopBP1 function.