Mature Epstein-Barr virus (EBV) was purified from the culture medium of infected lymphocytes made functionally conditional for Zta activation of lytic replication by an in-frame fusion with a mutant estrogen receptor. Proteins in purified virus preparations were separated by gradient gel electrophoresis and trypsin-digested; peptides were then analyzed by tandem hydrophobic chromatography, tandem MS sequencing, and MS scans. Potential peptides were matched with EBV and human gene ORFs. Mature EBV was mostly composed of homologues of proteins previously found in a herpes virion. However, EBV homologues to herpes simplex virus capsid-associated or tegument components UL7 (BBRF2), UL14 (BGLF3), and EBV BFRF1 were not significantly detected. Instead, probable tegument components included the EBV and ␥-herpesvirus-encoded BLRF2, BRRF2, BDLF2 and BKRF4 proteins. Actin was also a major tegument protein, and cofilin, tubulin, heat shock protein 90, and heat shock protein 70 were substantial components. EBV envelope glycoprotein gp350 was highly abundant, followed by glycoprotein gH, intact and furincleaved gB, gM, gp42, gL, gp78, gp150, and gN. BILF1 (gp64) and proteins associated with latent EBV infection were not detected in virions.lymphocyte ͉ proteomics ͉ virion ͉ herpes virus T he experiments reported here determine the protein composition of mature enveloped Epstein-Barr (EB) virus (EBV). EBV protein composition has not been systematically studied since the proteins of purified enveloped and deenveloped EBV were initially displayed on polyacrylamide gels. Except for glycoproteins, EB virion protein annotations have usually been based on DNA sequence homology to a characterized herpesvirus ORF, with verification for EBV in specific instances.EBV is a ␥1-herpesvirus. The ␥1-and ␥2-herpesvirus genomes are mostly collinear and share smaller conserved gene clusters with ␣-and ␤-herpesvirus genomes. Herpesvirus family-conserved genes mostly encode proteins that are important for DNA replication, virus morphogenesis, or virion composition. Conserved herpesvirus genes are known to encode 5 capsid proteins, 5 envelope proteins, and 10 tegument proteins in at least one herpesvirus (1, 2). Accordingly, EBV BcLF1, BDLF1, BFRF3, BORF1, and BBRF1 ORFs are likely to encode the major, minor, and smallest capsid proteins (MCP, mCP, and sCP, respectively); the mCP-binding protein (mCPBP); and portal. Furthermore, the EBV BPLF1, BOLF1, BVRF1, BGLF1, BGLF4, BGLF2, BBRF2, BSRF1, BGLF3, and BBLF1 ORFs are likely to encode tegument proteins (3-5). EBV BNRF1 and BLRF2 probably encode ␥-herpesvirus-unique tegument proteins (Table 1) (6-8). The EBV homologues of gB (BALF4), gH (BXLF2), and gL (BKRF2) have been detected by specific antibodies in EB virions. Moreover, EBV BLLF1, BZLF2, and BDLF3 ORF-specific antibodies have detected EBV-unique gp350, gp42, and gp150 in the virus (Table 1) (9). EBV BMRF2-encoded protein may also be in virus envelopes because it has an RGD (arginine-glycine-aspartic acid) motif that may be a ligand for an int...

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.

Lymphoblastoid cell lines (LCLs), originally collected as renewable sources of DNA, are now being used as a model system to study genotype–phenotype relationships in human cells, including searches for QTLs influencing levels of individual mRNAs and responses to drugs and radiation. In the course of attempting to map genes for drug response using 269 LCLs from the International HapMap Project, we evaluated the extent to which biological noise and non-genetic confounders contribute to trait variability in LCLs. While drug responses could be technically well measured on a given day, we observed significant day-to-day variability and substantial correlation to non-genetic confounders, such as baseline growth rates and metabolic state in culture. After correcting for these confounders, we were unable to detect any QTLs with genome-wide significance for drug response. A much higher proportion of variance in mRNA levels may be attributed to non-genetic factors (intra-individual variance—i.e., biological noise, levels of the EBV virus used to transform the cells, ATP levels) than to detectable eQTLs. Finally, in an attempt to improve power, we focused analysis on those genes that had both detectable eQTLs and correlation to drug response; we were unable to detect evidence that eQTL SNPs are convincingly associated with drug response in the model. While LCLs are a promising model for pharmacogenetic experiments, biological noise and in vitro artifacts may reduce power and have the potential to create spurious association due to confounding.