Viruses that establish latent infection must maintain their DNA in the host nucleus through many cellular generations. Here we identify a novel mechanism by which the gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) may achieve this persistence in latently infected body cavity-based lymphoma (BCBL) cells. We find that KSHV genomic DNA is associated with host chromosomes and colocalizes with the latency-associated nuclear antigen (LANA). Furthermore, a region at the left end of the KSHV genome binds strongly to LANA and can colocalize to the host chromosomes with LANA. Additionally, we found that LANA associates with histone H1 in KSHV-infected BCBL cells. We propose that this chromosomal association of the KSHV genome is mediated by LANA and involves a tethering mechanism by which viral episomes are linked to host chromatin through simultaneous interaction with host chromosomal proteins including histone H1 and cis-acting KSHV DNA elements. This strategy may be employed by other viruses in establishment of latency in the infected cells.

Epstein-Barr virus (EBV) is an oncogenic virus associated with a number of human malignancies including Burkitt lymphoma, nasopharyngeal carcinoma, lymphoproliferative disease and, though still debated, breast carcinoma. A subset of latent EBV antigens is required for mediating immortalization of primary B-lymphocytes. Here we demonstrate that the carboxy-terminal region of the essential latent antigen, EBNA-3C, interacts specifically with the human metastatic suppressor protein Nm23-H1. Moreover, EBNA-3C reverses the ability of Nm23-H1 to suppress the migration of Burkitt lymphoma cells and breast carcinoma cells. We propose that EBNA-3C contributes to EBV-associated human cancers by targeting and altering the role of the metastasis suppressor Nm23-H1.

Latent infection by members of the gammaherpesvirus family is typically characterized by stable episomal maintenance of genomic viral DNA. In the case of Epstein--Barr virus (EBV), this is dependent upon binding of the Epstein-Barr nuclear antigen 1 (EBNA1) to sites which lie within the origin of plasmid replication (OriP). The recently discovered Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the latency-associated nuclear antigen (LANA), which appears to be important for supporting the latent infection of human cells by KSHV. The present work describes site-specific binding of the LANA protein to multiple different elements at the left end of the genome, a region which appears to be critical for maintenance of KSHV episomes. Of the three sites, terminal LANA-binding region 4 (TLBR4) binds LANA with the highest affinity when compared to the other sites. Further characterization of this cis-acting element by mutagenesis studies indicates that the minimal TLBR4-binding sequence is represented by a 13-bp sequence 5prime prime or minute CGCCCGGGCATGG 3prime prime or minute. Furthermore, this specific binding to TLBR4 was mediated by the distal 200 amino acid C-terminus of the LANA protein.

Telomerase is a multi-subunit ribonucleoprotein holoenzyme that stabilizes telomere length through the addition of new repeat sequence to the ends of chromosomes. Telomerase reverse transcriptase is the subunit of this complex responsible for the enzymatic activity of telomerase. Expression of the reverse transcriptase is regulated at the level of transcription through the action of transcription factors that target its promoter. Most Kaposi's sarcoma tumor cells are latently infected with the Kaposi's sarcoma-associated herpesvirus, and the constitutive expression of a viralencoded latency-associated nuclear antigen has been shown to be important for the maintenance of the viral episome. The proliferative nature of Kaposi's sarcoma suggests that this antigen may also play a critical role in viral-mediated oncogenesis. In this study telomerase reverse transcriptase promoter elements cloned into a luciferase reporter plasmid were analyzed to determine the ability of the latency-associated nuclear antigen to regulate transcription. The latency-associated nuclear antigen transactivated the full-length promoter in 293T, 293, and BJAB cell lines. Furthermore, truncation promoter studies implicated sequence from ؊130 to ؉5 in viral-mediated activation. This region contains five Sp1 transcription factor-binding sites. Electrophoretic mobility shift assays indicated that the latency-associated nuclear antigen targets and affects the Sp1-DNA complex in the context of BJAB nuclear extracts.

The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for EBV-dependent immortalization of human primary B lymphocytes. Genetic analysis indicated that amino acids 365 to 992 are important for EBV-mediated immortalization of B lymphocytes. We demonstrate that this region of EBNA3C critical for immortalization interacts with prothymosin alpha (ProT␣), a cellular protein previously identified to be important for cell division and proliferation. This interaction maps to a region downstream of amino acid 365 known to be involved in transcription regulation and critical for EBV-mediated transformation of primary B lymphocytes. Additionally, we show that EBNA3C also interacts with p300, a cellular acetyltransferase. This interaction suggests a possible role in regulation of histone acetylation and chromatin remodeling. An increase in histone acetylation was observed in EBV-transformed lymphoblastoid cell lines, which is consistent with increased cellular gene expression. These cells express the entire repertoire of latent nuclear antigens, including EBNA3C. Expression of EBNA3C in cells with increased acetyltransferase activity mediated by the EBV transactivator EBNA2 results in down-modulation of this activity in a dose-responsive manner. The interactions of EBNA3C with ProT␣ and p300 provide new evidence implicating this essential EBV protein EBNA3C in modulating the acetylation of cellular factors, including histones. Hence, EBNA3C plays a critical role in balancing cellular transcriptional events by linking the biological property of mediating inhibition of EBNA2 transcription activation and the observed histone acetyltransferase activity, thereby orchestrating immortalization of EBV-infected cells.Epstein-Barr Virus (EBV) is a human gammaherpesvirus predominantly infecting epithelial cells of the oropharynx and human primary B lymphocytes (41,63). EBV is the etiological agent of infectious mononucleosis and is also associated with various human malignancies, including Burkitt's lymphoma, nasopharyngeal carcinoma, non-Hodgkin's disease, AIDS immunoblastic lymphomas, and lymphoproliferative disease (3,63). Infection of the oropharyngeal epithelium is predominantly a lytic type of infection with the production of progeny virus (33,61,63,73). Infection of human primary B lymphocytes by EBV transforms them into continuously proliferating lymphoblastoid cell lines (LCLs) in vitro (11,29). Recent studies have demonstrated that EBV utilizes two major cellular signaling pathways for transforming B cells, the NOTCH1 signaling pathway and the TNF signaling pathway (6, 34, 57).After initial infection of B lymphocytes, EBV typically establishes a latent infection with the expression of 11 viral transcripts (41, 63). These genes are the six EBV nuclear antigens (EBNAs), three latent membrane proteins (LMPs), and the EBV early RNAs (41). Only a selected number of these genes are necessary for EBV-mediated immortalization of B lymphocytes (65). EBNA2, EBNA3A, EBNA3C, and LMP1 are essential for EBV-induced immorta...

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Purpose: UV radiation is the major environmental risk factor for melanoma and a potent inducer of oxidative stress, which is implicated in the pathogenesis of several malignancies.We evaluated whether the thiol antioxidant N-acetylcysteine (NAC) could protect melanocytes from UVinduced oxidative stress/damage in vitro and from UV-induced melanoma in vivo. Experimental Design: In vitro experiments used the mouse melanocyte line melan-a. For in vivo experiments, mice transgenic for hepatocyte growth factor and survivin, shown previously to develop melanoma following a single neonatal dose of UV irradiation, were given NAC (7 mg/mL; mother's drinking water) transplacentally and through nursing until 2 weeks after birth. Results: NAC (1-10 mmol/L) protected melan-a cells from several UV-induced oxidative sequelae, including production of intracellular peroxide, formation of the signature oxidative DNA lesion 8-oxoguanine, and depletion of free reduced thiols (primarily glutathione). Delivery of NAC reduced thiol depletion and blocked formation of 8-oxoguanine in mouse skin following neonatal UV treatment. Mean onset of UV-induced melanocytic tumors was significantly delayed in NAC-treated compared with control mice (21versus 14 weeks; P = 0.0003). Conclusions: Our data highlight the potential importance of oxidative stress in the pathogenesis of melanoma and suggest that NAC may be useful as a chemopreventive agent.

The latency-associated nuclear antigen (LANA) encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) is expressed in the majority of KSHV-infected cells and in cells coinfected with Epstein-Barr virus (EBV). In coinfected body cavity-based lymphomas (BCBLs), EBV latent membrane protein 1 (LMP1), which is essential for B-lymphocyte transformation, is expressed. EBNA2 upregulates the expression of LMP1 and other cellular genes through specific interactions with cellular transcription factors tethering EBNA2 to its responsive promoters. In coinfected BCBL cells, EBNA2 is not detected but LANA, which is constitutively expressed, contains motifs suggestive of potential transcriptional activity. Additionally, recent studies have shown that LANA is capable of activating cellular promoters. Therefore, we investigated whether LANA can affect transcription from two major EBV latent promoters. In this study, we demonstrated that LANA can efficiently transactivate both the LMP1 and C promoters in the human B-cell line BJAB as well as in the human embryonic kidney 293 cell line. Moreover, we demonstrated that specific domains of LANA containing the putative leucine zipper and the glutamic acid-rich region are highly effective in upregulating these viral promoters, while the amino-terminal region (435 amino acids) exhibited little or no transactivation activity in our assays. We also specifically tested truncations of the LMP1 promoter element and showed that the ؊204 to ؉40 region had increased levels of activation compared with a larger region, ؊512 to ؉40, which contains two recombination signal-binding protein J binding sites. The smaller, ؊204 to ؉40 promoter region contains specific binding sites for the Ets family transcription factor PU.1, transcription activating factor/cyclic AMP response element, and Sp1, all of which are known to function as activators of transcription. Our data therefore suggest a potential role for LANA in regulation of the major EBV latent promoters in KSHV-and EBVcoinfected cells. Furthermore, LANA may be able to activate transcription of viral and cellular promoters in the absence of EBNA2, potentially through association with transcription factors bound to their cognate sequences within the ؊204 to ؉40 region. This regulation of viral gene expression is critical for persistence of these DNA tumor viruses and most likely involved in mediating the oncogenic process in these coinfected cells.