Erle S. Robertson

Kaposi's Sarcoma-Associated Herpesvirus-Encoded Latency-Associated Nuclear Antigen Inhibits Lytic Replication by Targeting Rta: a Potential Mechanism for Virus-Mediated Control of Latency

ABSTRACT Like other herpesviruses, Kaposis sarcoma-associated herpesvirus (KSHV, also designated human herpesvirus 8) can establish a latent infection in the infected host. During latency a small number of genes are expressed. One of those genes encodes latency-associated nuclear antigen (LANA), which is constitutively expressed in cells during latent as well as lytic infection. LANA has previously been shown to be important for the establishment of latent episome maintenance through tethering of the viral genome to the host chromosomes. Under specific conditions, KSHV can undergo lytic replication, with the production of viral progeny. The immediate-early Rta, encoded by open reading frame 50 of KSHV, has been shown to play a critical role in switching from viral latent replication to lytic replication. Overexpression of Rta from a heterologous promoter is sufficient for driving KSHV lytic replication and the production of viral progeny. In the present study, we show that LANA down-modulates Rtas promoter activity in transient reporter assays, thus repressing Rta-mediated transactivation. This results in a decrease in the production of KSHV progeny virions. We also found that LANA interacts physically with Rta both in vivo and in vitro. Taken together, our results demonstrate that LANA can inhibit viral lytic replication by inhibiting expression as well as antagonizing the function of Rta. This suggests that LANA may play a critical role in maintaining latency by controlling the switch between viral latency and lytic replication.

Oncogenic Forms of NOTCH1 Lacking Either the Primary Binding Site for RBP-Jκ or Nuclear Localization Sequences Retain the Ability to Associate with RBP-Jκ and Activate Transcription

Truncated forms of the NOTCH1 transmembrane receptor engineered to resemble mutant forms of NOTCH1 found in certain cases of human T cell leukemia/lymphoma (T-ALL) efficiently induce T-ALL when expressed in the bone marrow of mice. Unlike full-sized NOTCH1, two such truncated forms of the protein either lacking a major portion of the extracellular domain (ΔE) or consisting only of the intracellular domain (ICN) were found to activate transcription in cultured cells, presumably through RBP-Jκ response elements within DNA. Both truncated forms also bound to the transcription factor RBP-Jκ in extracts prepared from human and murine T-ALL cell lines. Transcriptional activation required the presence of a weak RBP-Jκ-binding site within the NOTCH1 ankyrin repeat region of the intracellular domain. Unexpectedly, a second, stronger RBP-Jκ-binding site, which lies within the intracellular domain close to the transmembrane region and significantly augments association with RBP-Jκ, was not needed for oncogenesis or for transcriptional activation. While ICN appeared primarily in the nucleus, ΔE localized to cytoplasmic and nuclear membranes, suggesting that intranuclear localization is not essential for oncogenesis or transcriptional activation. In support of this interpretation, mutation of putative nuclear localization sequences decreased nuclear localization and increased transcriptional activation by membrane-bound ΔE. Transcriptional activation by this mutant form of membrane-bound ΔE was approximately equivalent to that produced by intranuclear ICN. These data are most consistent with NOTCH1 oncogenesis and transcriptional activation being independent of association with RBP-Jκ at promoter sites.

EC5S ubiquitin complex is recruited by KSHV latent antigen LANA for degradation of the VHL and p53 tumor suppressors.

Cellular protein degradation pathways can be utilized by viruses to establish an environment that favors their propagation. Here we report that the Kaposis sarcoma–associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) directly functions as a component of the EC5S ubiquitin complex targeting the tumor suppressors von Hippel-Lindau (VHL) and p53 for degradation. We have characterized a suppressor of cytokine signaling box-like motif within LANA composed of an Elongin B and C box and a Cullin box, which is spatially located at its amino and carboxyl termini. This motif is necessary for LANA interaction with the Cul5–Elongin BC complex, to promote polyubiquitylation of cellular substrates VHL and p53 in vitro via its amino- and carboxyl-terminal binding domain, respectively. In transfected cells as well as KSHV-infected B lymphoma cells, LANA expression stimulates degradation of VHL and p53. Additionally, specific RNA interference–mediated LANA knockdown stabilized VHL and p53 in primary effusion lymphoma cells. Thus, manipulation of tumor suppressors by LANA potentially provides a favorable environment for progression of KSHV-infected tumor cells.

Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Up-Regulates Transcription of Human Telomerase Reverse Transcriptase Promoter through Interaction with Transcription Factor Sp1

ABSTRACT Telomerase is required for the maintenance of telomere length and is an important determinant for cell immortalization. In human cells, telomerase activity is due to the expression of its enzymatic subunit, human telomerase reverse transcriptase (hTERT). The expression of hTERT is not typically detectable in healthy somatic human cells but is present in cancerous tissues and immortalized cells. We have previously shown that hTERT promoter activity is up-regulated by the Kaposis sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA). LANA is expressed in all forms of human malignancies associated with KSHV. The hTERT promoter sequence located at positions −130 to +5 contains several Sp1 binding motifs and was shown be important for up-regulation by LANA. In this report, we demonstrate that hTERT promoter activity is due to the direct interaction of LANA with Sp1. The interaction of LANA with Sp1 was demonstrated through in vitro binding experiments and coimmunoprecipitation and is supported by the colocalization of these two molecules in the nuclei of KSHV-infected cells. Moreover, LANA modulates Sp1-mediated transcription in transient GAL4 fusion reporter assays. Mapping of the regions involved in binding and transcriptional activation showed that the amino terminus of LANA is the major site for interaction and up-regulation but that it can cooperate with the carboxy terminus to enhance these functions. An analysis of Sp1 binding to its cognate sequence corroborated the binding data. Together, our results suggest that the interaction of LANA with Sp1 up-regulates the telomerase promoter, potentially contributing to the immortalization of KSHV-infected cells.

Epstein-Barr virus nuclear protein EBNA-3C interacts with the human metastatic suppressor Nm23-H1: A molecular link to cancer metastasis

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.

Kaposi's sarcoma-associated herpesvirus reactivation is regulated by interaction of latency-associated nuclear antigen with recombination signal sequence-binding protein Jkappa, the major downstream effector of the Notch signaling pathway.

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is the major biological cofactor contributing to development of Kaposis sarcoma. KSHV establishes a latent infection in human B cells expressing the latency-associated nuclear antigen (LANA), a critical factor in the regulation of viral latency. LANA controls KSHV latent infection through repression of RTA, an activator of many lytic promoters. RTA activates the expression of several lytic viral genes by interacting with recombination signal sequence-binding protein Jκ (RBP-Jκ), a transcriptional repressor and the target of the Notch signaling pathway. The recognition that a number of KSHV lytic gene promoters, including RTA, contain RBP-Jκ binding sites raised the possibility that RBP-Jκ-mediated repression may be central to the establishment of latency. Here, we tested this hypothesis by examining the regulation of RTA by LANA through binding to RBP-Jκ. This study demonstrates that LANA physically associates with RBP-Jκ in vitro and in KSHV-infected cells, with the complex formed capable of binding to RBP-Jκ cognate sequences. RBP-Jκ binding sites within the RTA promoter have been found to be critical for LANA-mediated repression. Our study describes a novel mechanism through which LANA maintains KSHV latency by targeting a major downstream effector of the Notch signaling pathway.

Epstein-Barr Virus Nuclear Antigen 3C Recruits Histone Deacetylase Activity and Associates with the Corepressors mSin3A and NCoR in Human B-Cell Lines

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a known regulatory transcription factor that has been shown to interact with histone deacetylase 1 (HDAC1) when cotransfected in human cell lines and by in vitro binding experiments. Previous studies have shown that EBNA3C interacts with p300 and prothymosin alpha (ProTα) in EBV-infected cells and may be involved in recruiting acetyltransferases to the chromatin for acetylation of histones and transcriptional activation. EBNA3C has also been shown to function as a repressor of transcription when directed to promoters. In this report, we show that EBNA3C complexed with ProTα can also recruit deacetylase activity and associates in a complex that includes HDAC1 and HDAC2 in human B cells. A complex of EBNA3C and ProTα coimmunoprecipitated with HDAC1 and HDAC2 in cell lines stably expressing EBNA3C. Additionally, this complex associated with the mSin3A and NCoR corepressors in EBNA3C-expressing cell lines and may function in a complex with additional transcription factors known to be repressors of transcription. EBNA3C in complex with ProTα recruited deacetylase activity in cell lines stably expressing EBNA3C, and this activity was shown to be partially sensitive to trichostatin A (TSA). This suggests an association with other deacetylases that are insensitive to the general inhibitory effects of TSA, as the entire activity was not abolished in multiple assays. The association between EBNA3C and the corepressors as well as HDACs is likely to depend on the presence of ProTα in the complex. Immunoprecipitation with anti-ProTα antibody immunoprecipitated EBNA3C and the other repressors, whereas immunoprecipitation with anti-EBNA3C antibody resulted in little or no association with these molecules associated with transcription repression. Clearly, EBNA3C functions as a component of a number of dynamic complexes which function in repression and activation of transcription.

Structure and Function of Latency-Associated Nuclear Antigen

Latency-associated nuclear antigen (LANA) encoded by open reading frame 73 (ORF73) is the major latent protein expressed in all forms of KSHV-associated malignancies. LANA is a large (222-234 kDa) nuclear protein that interacts with various cellular as well as viral proteins. LANA has been classified as an oncogenic protein as it dysregulates various cellular pathways including tumor suppressor pathways associated with pRb and p53 and can transform primary rat embryo fibroblasts in cooperation with the cellular oncogene Hras. It associates with GSK-3beta, an important modulator of Wnt signaling pathway leading to the accumulation of cytoplasmic beta-catenin, which upregulates Tcf/Lef regulated genes after entering into the nucleus. LANA also blocks the expression of RTA, the reactivation transcriptional activator, which is critical for the latency to lytic switch, and thus helps in maintaining viral latency. LANA tethers the viral episomal DNA to the host chromosomes by directly binding to its cognate binding sequence within the TR region of the genome through its C terminus and to the nucleosomes through the N terminus of the molecule. Tethering to the host chromosomes helps in efficient partitioning of the viral episomes in the dividing cells. Disruptions of LANA expression led to reduction in the episomal copies of the viral DNA, supporting its role in persistence of the viral DNA. The functions known so far suggest that LANA is a key player in KSHV-mediated pathogenesis.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded Latency-Associated Nuclear Antigen Induces Chromosomal Instability through Inhibition of p53 Function

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is predominantly associated with three human malignancies, KS, primary effusion lymphoma, and multicentric Castlemans disease. These disorders are linked to genomic instability, known to be a crucial component of the oncogenic process. Latency-associated nuclear antigen (LANA), encoded by open reading frame 73 of the KSHV genome, is a latent protein consistently expressed in all KSHV-associated diseases. LANA is important in viral genome maintenance and is associated with cellular and viral proteins to regulate viral and cellular gene expression. LANA interacts with the tumor suppressor genes p53 and pRb, indicating that LANA may target these proteins and promote oncogenesis. In this study, we generated cell lines which stably expressed LANA to observe the effects of LANA expression on cell phenotype. LANA expression in these stable cell lines showed a dramatic increase in chromosomal instability, indicated by the presence of increased multinucleation, micronuclei, and aberrant centrosomes. In addition, these stable cell lines demonstrated an increased proliferation rate and as well as increased entry into S phase in both stable and transiently transfected LANA-expressing cells. Additionally, p53 transcription and its transactivation activity were suppressed by LANA expression in a dose-dependent manner. LANA may therefore promote chromosomal instability by suppressing the functional activities of p53, thereby facilitating KSHV-mediated pathogenesis and cancer.

Induction of Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen by the Lytic Transactivator RTA: a Novel Mechanism for Establishment of Latency

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is the etiological agent contributing to development of Kaposis sarcoma, primary effusion lymphoma, and multicentric Castleman desease. Following primary infection, latency is typically established. However, the mechanism by which KSHV establishes latency is not understood. We have reported that the latency-associated nuclear antigen (LANA) can repress RTA (for replication and transcription activator) expression by down-regulating its promoter. In this study, we show that RTA is associated with the virion particle. We also show that RTA can activate the LANA promoter and induce LANA expression in transient reporter assays. Additionally, the transcription of RTA correlates with LANA expression in the early stages of de novo infection of KSHV, and induction of LANA transcription is responsive to induction of RTA with an inducible system. This induction in LANA transcription was dependent on recombination signal sequence binding protein Jκ (RBP-Jκ), as a RBP-Jκ-deficient cell line was significantly delayed and inefficient in LANA transcription with expression of RTA. These studies suggest that RTA contributes to establishment of KSHV latency by activating LANA expression in the early stages of infection by utilizing the major effector of the Notch signaling pathway RBP-Jκ. This describes a feedback mechanism by which LANA and RTA can regulate each other and is likely to be a key event in the establishment of KSHV latency.