The natural history of cancers associated with virus exposure is intriguing, since only a minority of human tissues infected with these viruses inevitably progress to cancer. However, the molecular reasons why the infection is controlled or instead progresses to subsequent stages of tumorigenesis are largely unknown. In this article, we provide the first complete DNA methylomes of double-stranded DNA viruses associated with human cancer that might provide important clues to help us understand the described process. Using bisulfite genomic sequencing of multiple clones, we have obtained the DNA methylation status of every CpG dinucleotide in the genome of the Human Papilloma Viruses 16 and 18 and Human Hepatitis B Virus, and in all the transcription start sites of the Epstein-Barr Virus. These viruses are associated with infectious diseases (such as hepatitis B and infectious mononucleosis) and the development of human tumors (cervical, hepatic, and nasopharyngeal cancers, and lymphoma), and are responsible for 1 million deaths worldwide every year. The DNA methylomes presented provide evidence of the dynamic nature of the epigenome in contrast to the genome. We observed that the DNA methylome of these viruses evolves from an unmethylated to a highly methylated genome in association with the progression of the disease, from asymptomatic healthy carriers, through chronically infected tissues and pre-malignant lesions, to the full-blown invasive tumor. The observed DNA methylation changes have a major functional impact on the biological behavior of the viruses.

Epstein-Barr virus (EBV) is a human herpesvirus hiding in a latent form in memory B cells in the majority of the world population. Although, primary EBV infection is asymptomatic or causes a self-limiting disease, infectious mononucleosis, the virus is associated with a wide variety of neoplasms developing in immunosuppressed or immunodeficient individuals, but also in patients with an apparently intact immune system. In memory B cells, tumor cells, and lymphoblastoid cell lines (LCLs, transformed by EBV in vitro) the expression of the viral genes is highly restricted. There is no virus production (lytic viral replication associated with the expression of all viral genes) in tight latency. The expression of latent viral oncogenes and RNAs is under a strict epigenetic control via DNA methylation and histone modifications that results either in a complete silencing of the EBV genome in memory B cells, or in a cell-type dependent usage of latent promoters in tumor cells, germinal center B cells, and LCLs. Both the latent and lytic EBV proteins are potent immunogens and elicit vigorous B- and T-cell responses. In immunosuppressed and immunodeficient patients, or in individuals with a functional defect of EBV-specific T cells, lytic EBV replication is regularly activated and an increased viral load can be detected in the blood. Enhanced lytic replication results in new infection events and EBV-associated transformation events, and seems to be a risk factor both for malignant transformation and the development of autoimmune diseases. One may speculate that an increased load or altered presentation of a limited set of lytic or latent EBV proteins that cross-react with cellular antigens triggers and perpetuates the pathogenic processes that result in multiple sclerosis, systemic lupus erythematosus (SLE), and rheumatoid arthritis. In addition, in SLE patients EBV may cause defects of B-cell tolerance checkpoints because latent membrane protein 1, an EBV-encoded viral oncoprotein can induce BAFF, a B-cell activating factor that rescues self-reactive B cells and induces a lupus-like autoimmune disease in transgenic mice.

A method, using HPLC combined with electrospray tandem mass spectrometry (ES-MS/MS), was developed and validated to detect and quantify the major DNA adduct resulting from exposure to the ultimate tumorigenic benzo[a]pyrene (BP) metabolite, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Calf thymus DNA was reacted with BPDE, digested enzymatically to nucleosides, and the major DNA adduct, 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-BPDE), was purified by HPLC. Similar procedures were applied to prepare dG-BPDE-d8 from [1,2,3,4,5,6,11,12-(2)H8]BPDE for use as an internal standard. The HPLC-ES-MS/MS method was validated using a mixture of hydrolyzed salmon testis DNA (82 microg) and 10 pg dG-BPDE (analogous to 6.9 adducts/10(8) nucleotides). The results indicated an inter- and intraday accuracy of 99-100% and precision of 1.6-1.7% (relative standard deviation). When applied to a calf thymus DNA sample modified in vitro with [1,3-(3)H]BPDE, the method gave a value very similar to those obtained by radiolabeling, (32)P-postlabeling, and immunoassay. HPLC-ES-MS/MS analysis of hepatic DNA from mice treated intraperitoneally with 0.5 and 1.0 mg of [7,8-(3)H]BP gave values comparable to those determined by 32P-postlabeling and immunoassay. Lung DNA from mice fed a 0.3% coal tar diet (containing approximately 2 mg BP/g coal tar) for one month had 0.6 +/- 0.04 dG-BPDE adducts/10(8) nucleotides. This value is much lower than the 102 +/- 14 total DNA adducts/10(8) nucleotides determined by 32P-postlabeling, which suggests that dG-BPDE makes only a minor contribution to the DNA adducts formed in lung tissue of mice administered coal tar. The HPLC-ES-MS/MS method was used to assess human lung DNA samples for the presence of dG-BPDE. Based upon a limit of detection of 0.3 dG-BPDE adducts/10(8) nucleotides, when using 100 microg of DNA, dG-BPDE was detected in only 1 out of 26 samples. These observations indicate that HPLC-ES-MS/MS is suitable to assess the contribution of BP to DNA damage caused by exposures to polycyclic aromatic hydrocarbon (PAH) mixtures. The results further suggest that dG-BPDE may contribute only a small fraction of the total DNA adducts detected by other DNA adduct methodologies in individuals exposed to PAHs.

Three alkaloids, lycorine, homolycorine and 2- O-acetyllycorine, were isolated from the bulbs of Leucojum vernum (Amaryllidaceae) and identified by means of NMR analysis. The alkaloids obtained from L. vernum and from other Amaryllidaceae species were studied in vitro for HIV-1 replication inhibitory activity on MT4 cells. The cytotoxicity of the compounds in uninfected cells was evaluated by using the MTT assay and the [ (3)H]thymidine incorporation test. The antiviral activities were determined by means of the p24 antigen assay and solid-phase reverse transcriptase testing. The results demonstrate that trisphaeridine, lycorine, homolycorine, and haemanthamine possess high antiretroviral activities (IC (50) = 0.4 - 7.3 microg/mL), accompanied by low therapeutic indices (TI (50) = 1.3 - 1.9).

SUMMARYSeven virus-encoded proteins are regularly expressed in Epstein-Barr virus (EBV)-transformed lymphoblastoid (LCL) cell lines: the EBV nuclear antigens EBNA 1 to 6 and the latent membrane protein (LMP). In nasopharyngeal carcinoma (NPC), only EBNA 1 is regularly expressed; LMP is detected in about 50~ of the tumours. In Burkitt's lymphoma (BL) tumours, only EBNA 1 is expressed. Also, in BL-derived cell lines that maintain the phenotypic markers characteristic of the in vivo tumour (group I), only EBNA 1 is expressed. EBV was rescued by induction or cocultivation from one BL cell line with a restricted group I pattern, and from one NPC tumour, into normal B cells. In the resulting LCLs EBNA 1 to 6 and LMP were expressed. We assessed the level of methylation in the genes encoding EBNA 2 and LMP by restriction fragment analysis using the methylation-sensitive enzymes SmaI and HpalI. These genes were extensively methylated in the group I BL line Rael and the nude mouse-passaged C15 NPC tumour, but were demethylated in the derived LCLs. In the LMP expressing the NPC tumour, but were demethylated in the derived LCLs. In the LMP-expressing coding exons were methylated. The EBNA 1 coding exon was methylated in the Rael line and in NPC, in spite of expression. In contrast, CpG pairs in ori P were originally hypomethylated and remained so after their transfer to LCLs. The cell phenotypedependent pattern of EBV gene methylation correlated with the phenotype-dependent pattern of EBNA and LMP expression. The specific patterns of methylation localized to controlling regions (ori P and 5' flanking sequences) also suggest a specific role for methylation in the regulation of EBNA and LMP expression.

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