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...

Polyploidy is often an early event during cervical carcinogenesis, and it predisposes cells to aneuploidy, which is thought to play a causal role in tumorigenesis. Cervical and anogenital cancers are induced by the high-risk types of human papillomavirus (HPV). The HPV E6 oncoprotein induces polyploidy in human keratinocytes, yet the mechanism is not known. It was believed that E6 induces polyploidy by abrogating the spindle checkpoint after mitotic stress. We have tested this hypothesis using human keratinocytes in which E6 expression induces a significant amount of polyploidy. We found that E6 expression does not affect the spindle checkpoint. Instead, we provide direct evidence that E6 is capable of abrogating the subsequent G 1 arrest after adaptation of the mitotic stress. E6 targets p53 for degradation, and previous studies have shown an important role for p53 in modulation of the G 1 arrest after mitotic stress. Importantly, we have discovered that E6 mutants defective in p53 degradation also induce polyploidy, although with lower efficiency. These results suggest that E6 is able to induce polyploidy via both p53-dependent and p53-independent mechanisms. Therefore, our studies highlight a novel function of HPV E6 that may contribute to HPV-induced carcinogenesis and improve our understanding of the onset of the disease. [Cancer Res 2007;67(6):2603-10]

To evaluate the role of Epstein-Barr Virus (EBV) nuclear antigen 3A (EBNA3A) in the continuous proliferation of EBV-infected primary B lymphocytes as lymphoblastoid cell lines (LCLs), we derived LCLs that are infected with a recombinant EBV genome that expresses EBNA3A fused to a 4-hydroxy-tamoxifen (4HT)-dependent mutant estrogen receptor hormone binding domain (EBNA3AHT). The LCLs grew similarly to wild-type LCLs in medium with 4HT despite a reduced level of EBNA3AHT fusion protein expression. In the absence of 4HT, EBNA3AHT moved from the nucleus to the cytoplasm and was degraded. EBNA3AHT-infected LCLs were unable to grow in medium without 4HT. The precise time to growth arrest varied inversely with cell density. Continued maintenance in medium without 4HT resulted in cell death, whereas readdition of 4HT restored cell growth. Expression of other EBNAs and LMP1, of CD23, and of c-myc was unaffected by EBNA3A inactivation. Wild-type EBNA3A expression from an oriP plasmid transfected into the LCLs protected the EBNA3AHT-infected LCLs from growth arrest and death in medium without 4HT, whereas EBNA3B or EBNA3C expression was unable to protect the LCLs from growth arrest and death. These experiments indicate that EBNA3A has a unique and critical role for the maintenance of LCL growth and ultimately survival. The EBNA3AHT-infected LCLs are also useful for genetic and biochemical analyses of the role of EBNA3A domains in LCL growth.