Epstein-Barr virus nuclear antigen 3C interacts with and enhances the stability of the c-Myc oncoprotein.

Epstein-Barr virus (EBV) was the first human DNA virus to be associated with cancer. Its oncogenic potential was further demonstrated by its ability to transform primary B lymphocytes in vitro. EBV nuclear antigen 3C (EBNA3C) is one of a small subset of latent antigens critical for the transformation of human primary B lymphocytes. Although EBNA3C has been shown to modulate several cellular functions, additional targets involved in cellular transformation remain to be explored. EBNA3C can recruit key components of the SCF(Skp2) ubiquitin ligase complex. In this report, we show that EBNA3C residues 130 to 190, previously shown to bind to the SCF(Skp2) complex, also can strongly associate with the c-Myc oncoprotein. Additionally, the interaction of EBNA3C with c-Myc was mapped to the region of c-Myc that includes the highly conserved Skp2 binding domain. Skp2 has been shown to regulate c-Myc stability and also has been shown to function as a coactivator of transcription for c-Myc target genes. We now show that the EBV latent oncoprotein EBNA3C can stabilize c-Myc and that the recruitment of both c-Myc and its cofactor Skp2 to c-Myc-dependent promoters can enhance c-Myc-dependent transcription. This same region of EBNA3C also recruits and modulates the activity of retinoblastoma and p27, both major regulators of the mammalian cell cycle. The inclusion of c-Myc in the group of cellular targets modulated by this domain further accentuates the importance of these critical residues of EBNA3C in bypassing the cell cycle checkpoints.

Molecular biology of EBV in relationship to AIDS-associated oncogenesis.

Epstein-Barr virus (EBV) is a gammaherpesvirus of the Lymphocryptovirus genus, which infects greater than 90% of the world's population. Infection is nonsymptomatic in healthy individuals, but has been associated with a number of lymphoproliferative disorders when accompanied by immunosuppression. Like all herpesviruses, EBV has both latent and lytic replication programs, which allows it to evade immune clearance and persist for the lifetime of the host. Latent infection is characterized by replication of the viral genome as an integral part of the host cell chromosomes, and the absence of production of infectious virus. A further layer of complexity is added in that EBV can establish three distinct latency programs, in each of which a specific set of viral antigens is expressed. In most malignant disorders associated with EBV, the virus replicates using one of these three latency programs. In the most aggressive latency program, only 11 of the hitherto 85 identified open reading frames in the EBV genome are expressed. The other two latency programs express even smaller subsets of this repertoire of latent genes. The onset of the AIDS pandemic and the corresponding increase in individuals with acquired immunodeficiency resulted in a sharp increase in EBV-mediated AIDS-associated malignancies. This has sparked a renewed interest in EBV biology and pathogenesis.

Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus recruits uracil DNA glycosylase 2 at the terminal repeats and is important for latent persistence of the virus.

Latency-associated nuclear antigen (LANA) of KSHV is expressed in all forms of Kaposi's sarcoma-associated herpesvirus (KSHV)-mediated tumors and is important for TR-mediated replication and persistence of the virus. LANA does not exhibit any enzymatic activity by itself but is critical for replication and maintenance of the viral genome. To identify LANA binding proteins, we used a LANA binding sequence 1 DNA affinity column and determined the identities of a number of proteins associated with LANA. One of the identified proteins was uracil DNA glycosylase 2 (UNG2). UNG2 is important for removing uracil residues yielded after either misincorporation of dUTP during replication or deamination of cytosine. The specificity of the 'LANA-UNG2 interaction was confirmed by using a scrambled DNA sequence affinity column. Interaction of LANA and UNG2 was further confirmed by in vitro binding and coimmunoprecipitation assays. Colocalization of these proteins was also detected in primary effusion lymphoma (PEL) cells, as well as in a cotransfected KSHV-negative cell line. UNG2 binds to the carboxyl terminus of LANA and retains its enzymatic activity in the complex. However, no major effect on TR-mediated DNA replication was observed when a UNG2-deficient (UNG(-/-)) cell line was used. Infection of UNG(-/-) and wild-type mouse embryonic fibroblasts with KSHV did not reveal any difference; however, UNG(-/-) cells produced a significantly reduced number of virion particles after induction. Interestingly, depletion of UNG2 in PEL cells with short hairpin RNA reduced the number of viral genome copies and produced infection-deficient virus.

KSHV encoded LANA upregulates Pim-1 and is a substrate for its kinase activity.

Pim kinases are proto-oncogenes that are upregulated in a number of B cell cancers, including Epstein-Barr Virus (EBV) associated Burkitt's lymphoma. They have also been shown to be upregulated in Kaposi sarcoma-associated herpes virus (KSHV) infected primary B cells. Most cells in KSHV-associated tumors are latently infected and express only a small subset of viral genes, with KSHV latency associated nuclear antigen (LANA) being constitutively expressed. LANA regulates the transcription of a large number of cellular and viral genes. Here, we show that LANA upregulates transcription from the Pim-1 promoter (pPim-1) and map this activation to a region in the promoter located within the sequence (-681 to +37). We show that LANA expressing cells can proliferate faster and are better protected from drug induced apoptosis. Since transition through cell cycle check points and anti-apoptosis are functions associated with Pim-1, it is likely that higher Pim-1 expression in cells expressing LANA is responsible, at least in part, for this effect. A Pim-1 phosphorylation site was also identified within the amino-terminal domain of LANA. Using in vitro kinase assays, we confirmed that LANA was indeed a Pim-1 substrate, and the failure of Pim-1 to phosphorylate LANA mutated at SS205/6RR identified this site as the specific serine residues phosphorylated by Pim-1. This report provides valuable insight into yet another cellular signaling pathway subverted by KSHV LANA and suggests a contribution to KSHV related oncogenesis.

Efficient gene transfer to human epidermal keratinocytes on fibronectin: in vitro evidence for transduction of epidermal stem cells.

The epidermis is an attractive target for gene therapy because it is easily accessible and has great potential as an ectopic site for protein delivery in vivo. Genetically modified keratinocytes can be expanded in culture and used to generate three-dimensional skin equivalents, which can deliver therapeutic proteins either locally or systemically for the treatment of wounds or systemic diseases. Here we present an optimum protocol that yields consistently high retroviral gene transfer on a substrate of recombinant fibronectin (rFN). Gene transfer on rFN depends strongly on virus concentration and the density of target cells. Interestingly, the kinetics of gene transfer varies depending upon the origin--mouse or human--of virus-producer cells. Most notably, long-term growth and clonogenic assays show that transduction on rFN promotes gene transfer to epidermal stem cells and prevents loss of clonogenic potential due to exposure of cells to retroviral supernatant. In contrast, the traditional protocol transduces mostly differentiated keratinocytes. We also show that skin equivalents prepared from genetically modified keratinocytes display high levels of transgene expression, mainly in the suprabasal layers. Our results are important for cutaneous gene therapy and for biological studies that require efficient and permanent genetic modification.