Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is one of the essential latent antigens for primary B-cell transformation. Previous studies established that EBNA3C facilitates degradation of several vital cell cycle regulators, including the retinoblastoma (pRb) and p27KIP proteins, by recruitment of the SCF Skp2 E3 ubiquitin ligase complex. EBNA3C was also shown to be ubiquitinated at its N-terminal residues. Furthermore, EBNA3C can bind to and be degraded in vitro by purified 20S proteasomes. Surprisingly, in lymphoblastoid cell lines, EBNA3C is extremely stable, and the mechanism for this stability is unknown. In this report we show that EBNA3C can function as a deubiquitination enzyme capable of deubiquitinating itself in vitro as well as in vivo. Functional mapping using deletion and point mutational analysis showed that both the N-and C-terminal domains of EBNA3C contribute to the deubiquitination activity. We also show that EBNA3C efficiently deubiquitinates Mdm2, an important cellular proto-oncogene, which is known to be overexpressed in several human cancers. The data presented here further demonstrate that the N-terminal domain of EBNA3C can bind to the acidic domain of Mdm2. Additionally, the N-terminal domain of EBNA3C strongly stabilizes Mdm2. Importantly, EBNA3C simultaneously binds to both Mdm2 and p53 and can form a stable ternary complex; however, in the presence of p53 the binding affinity of Mdm2 toward EBNA3C was significantly reduced, suggesting that p53 and Mdm2 might share a common overlapping domain of EBNA3C. We also showed that EBNA3C enhances the intrinsic ubiquitin ligase activity of Mdm2 toward p53, which in turn facilitated p53 ubiquitination and degradation. Thus, manipulation of the oncoprotein Mdm2 by EBNA3C potentially provides a favorable environment for transformation and proliferation of EBV-infected cells.
We used a combination of mathematical modeling and experiments to investigate the rate-limiting steps of retroviral transduction on surface-bound fibronectin (FN) and identify the conditions that maximize the efficiency of gene transfer. Our results show that fibronectin-assisted gene transfer (FAGT) is a strong function of the time and temperature of virus incubation in FN-coated plates. Gene transfer increases sharply at short times, reaches a maximum at intermediate times, and eventually declines as a result of loss of retroviral activity. The maximum transduction efficiency and the time at which this is attained increase with decreasing temperature of virus incubation. Depending on the temperature and the type of target cells, the initial rate of gene transfer increases by 3- to 10-fold and the maximum transduction efficiency increases by 2- to 4-fold as compared to traditional transduction (TT). Interestingly, Polybrene (PB) inhibits FAGT in a dose-dependent manner by inhibiting binding of retrovirus to FN. In contrast to traditional transduction, FAGT yields higher than 10-fold transduction efficiencies with concentrated retrovirus stocks. Gene transfer is directly proportional to the concentration of the virus-containing medium with no sign of saturation for the range of concentrations tested. These results suggest that immobilization of recombinant retrovirus can be rationally optimized to yield high efficiency of gene transfer to primary cells and improve the prospect of gene therapy for the treatment of human disease.
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.
Latency-associated nuclear antigen (LANA) of KSHV is expressed in all forms of Kaposi's sarcomaassociated 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.
Primary effusion lymphoma (PEL) is a common cancer in AIDS patients closely associated with Kaposi’s sarcoma-associated herpesvirus (KSHV). Previously, we showed that KSHV latency associated nuclear antigen (LANA) stabilizes intracellular activated Notch1 (ICN) involved in maintenance of the malignant phenotype of KSHV infected PEL cells in vitro. The γ-secretase inhibitor (GSI) which specifically blocks the production of ICN slows down the proliferation of the KSHV infected PEL cell lines BCBL1, BC3 as well as JSC1 in vitro. In this study, we extended these studies to explore the possibility that manipulation of the Notch signaling by GSI would prevent the growth of the PEL tumors in vivo. We observed that the onset of tumorigenesis of KSHV infected PELs was significantly delayed in GSI treated SCID mice harboring the PEL cell lines. We also found that GSI treatment resulted in necrosis as well as apoptosis in tumors generated by the xenotransplanted KSHV positive PEL cell lines. In contrast, GSI had no effect on mice harboring BJAB cells, a KSHV negative Burkitt’s lymphoma cell line where ICN levels were negligible. Our study provides further evidence to suggest that targeted downregulation of abnormal Notch signaling has therapeutic potential for KSHV related primary effusion lymphomas.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.