Taegun Seo

Viral Interferon Regulatory Factor 1 of Kaposi's Sarcoma-Associated Herpesvirus Binds to p53 and Represses p53-Dependent Transcription and Apoptosis

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is related to the development of Kaposis sarcoma. Open reading frame K9 of KSHV encodes viral interferon regulatory factor 1 (vIRF1), which functions as a repressor of interferon- and IRF1-mediated signal transduction. In addition, vIRF1 acts as an oncogene to induce cellular transformation. Here we show that vIRF1 directly associates with the tumor suppressor p53 and represses its functions. The vIRF1 interaction domains of p53 are the DNA binding domain (amino acids [aa] 100 to 300) and the tetramerization domain (aa 300 to 393). p53 interacts with the central region (aa 152 to 360) of vIRF1. vIRF1 suppresses p53-dependent transcription and deregulates its apoptotic activity. These results suggest that vIRF1 may regulate cellular function by inhibiting p53.

Viral Interferon Regulatory Factor 1 of Kaposi's Sarcoma-Associated Herpesvirus Interacts with a Cell Death Regulator, GRIM19, and Inhibits Interferon/Retinoic Acid-Induced Cell Death

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) plays a significant role in the development of Kaposis sarcoma, primary effusion lymphoma, and some forms of multicentric Castlemans disease. The KSHV open reading frame K9 encodes the viral interferon (IFN) factor 1 (vIRF1), which downregulates IFN- and IRF-mediated transcriptional activation, and leads to cellular transformation in rodent fibroblasts and induction of tumors in nude mice. Using the yeast two-hybrid assay, we identified genes associated with retinoid-IFN-induced mortality-19 (GRIM19), which interacts directly with vIRF1, both in vivo and in vitro. The N-terminal region of vIRF1 is required for binding GRIM19. Colocalization of vIRF1 and GRIM19 was observed in 293T cells. The vIRF1 protein deregulates GRIM19-induced apoptosis in the presence of IFN/all-trans-retinoic acid (RA) and inhibits IFN/RA-induced cell death. Another DNA tumor viral protein, human papillomavirus type 16 E6, also binds GRIM19, suggesting that this is a general target of viral proteins. Our results collectively indicate that vIRF1 modulates IFN/RA-cell death signals via interactions with GRIM19.

The K-bZIP Protein from Kaposi's Sarcoma-Associated Herpesvirus Interacts with p53 and Represses Its Transcriptional Activity

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been implicated in the pathogenesis of Kaposis sarcoma. KSHV encodes K-bZIP (open reading frame K8), a protein that belongs to the basic region-leucine zipper (bZIP) family of transcription factors. Here we show that K-bZIP associates with the cellular transcription factor p53 directly in vitro and in vivo. This interaction requires the bZIP domain of K-bZIP and the carboxy-terminal region (amino acids 300 to 393) of p53. We also show that K-bZIP represses the transcriptional activity of p53 which is required for apoptosis of the host cell. These results imply that K-bZIP blocks p53-mediated host cell death through its interaction with p53.

Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus functionally interacts with heterochromatin protein 1

Latency-associated nuclear antigen (LANA) of Kaposis sarcoma-associated herpesvirus plays an important role in maintenance of the viral genome during latent infection. LANA additionally participates in the transcriptional regulation of several viral and cellular promoters. When tethered to constitutively active promoters, the protein exhibits transcriptional repressor activity. In this report, we further characterized cell type-, promoter-, and domain-specific transcriptional repression by LANA. We additionally speculated on the mechanism underlying transcriptional repression by the C terminus of the protein. Subnuclear localization patterns and association with heterochromatin suggested a possible link between LANA and heterochromatin protein 1, a representative heterochromatin-associated protein. In vivo and in vitro binding and immunofluorescence assays revealed that LANA associates with heterochromatin protein 1 in an isotype-specific manner. Furthermore, biochemical fractionation and transient replication assays supported the possibility that this interaction contributes to transcriptional repression, targeting to subnuclear structures, and latent DNA replication activity of LANA.

Inhibition of nuclear factor κB activity by viral interferon regulatory factor 3 of Kaposi's sarcoma-associated herpesvirus

Nuclear factor-κB (NF-κB) is a transcription factor that plays an important role in the immune system and cell death. Many viral proteins modulate NF-κB to escape host immune surveillance, promote cell survival, and enhance viral replication. In the present study, we show that NF-κB activity is downmodulated by viral interferon regulatory factor 3 (vIRF3), which is encoded by Kaposis sarcoma-associated herpesvirus open-reading frame K10.5. vIRF3 repressed NF-κB-dependent transcription in a dose-dependent manner and inhibited the activation of NF-κB induced by tumor necrosis factor (TNF)-α. In vivo studies showed vIRF3 inhibited IκB kinase β (IKKβ) activity, but not IKKα activity, resulting in reduced IκB phosphorylation. Immunofluorescence assays showed that vIRF3 interfered with nuclear translocation of NF-κB. In addition, consistent with the inhibition of NF-κB activity, vIRF3 sensitized cells to TNF-α-induced apoptosis. While vIRF3 interacts with IKKβ in vitro and in 293T cells, we were unable to demonstrate vIRF3–IKKβ interaction in BCBL-1 cells. Our results indicate that vIRF3 can regulate the host immune system and apoptosis via inhibition of NF-κB activity.

Kaposi's Sarcoma–Associated Herpesvirus Viral IFN Regulatory Factor 1 Inhibits Transforming Growth Factor-β Signaling

Kaposi9s sarcoma–associated herpesvirus, also called human herpesvirus 8, has been implicated in the pathogenesis of Kaposi9s sarcoma, body cavity–based primary effusion lymphoma, and some forms of multicentric Castleman9s disease. The Kaposi9s sarcoma–associated herpesvirus open reading frame K9 encodes viral IFN regulatory factor 1 (vIRF1), which functions as a repressor of IFN-mediated signal transduction. vIRF1 expression in NIH 3T3 cells leads to transformation and consequently induces malignant fibrosarcoma in nude mice, suggesting that vIRF1 is a strong oncoprotein. Here, we show that vIRF1 inhibited transforming growth factor-β (TGF-β) signaling via its targeting of Smad proteins. vIRF1 suppressed TGF-β-mediated transcription and growth arrest. vIRF1 directly interacted with both Smad3 and Smad4, resulting in inhibition of their transactivation activity. Studies using vIRF1 deletion mutants showed that the central region of vIRF1 was required for vIRF1 association with Smad3 and Smad4 and that this region was also important for inhibition of TGF-β signaling. In addition, we found that vIRF1 interfered with Smad3-Smad4 complex formation and inhibited Smad3/Smad4 complexes from binding to DNA. These results indicate that vIRF1 inhibits TGF-β signaling via interaction with Smads. In addition, the data indicate the TGF-β pathway is an important target for viral oncoproteins.

Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) open reading frame 36 protein is a serine protein kinase.

Kaposis sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that is implicated in the pathogenesis of Kaposis sarcoma. The nucleotide sequence of the KSHV open reading frame (ORF) 36 predicts a polypeptide with significant sequence homology to known protein kinases. In this paper, we show that KSHV ORF36 mRNA is expressed during lytic growth and that ORF36 protein is localized in the nucleus. To determine whether the KSHV ORF36 protein is a protein kinase, we expressed it as a glutathione S-transferase (GST) fusion protein (GST-ORF36). Affinity-purified preparations of the GST-ORF36 fusion protein revealed that the protein is autophosphorylated. Mutation of lysine-108 to glutamine dramatically decreased the protein kinase activity of the purified protein, supporting the hypothesis that the protein kinase activity is inherent to the ORF36 protein. Phosphoamino acid analysis showed that the KSHV ORF36 fusion protein is phosphorylated on a serine residue, implying that KSHV ORF36 encodes a serine protein kinase.

Characterization of Monoclonal Antibodies Against GRIM-19, a Novel IFN-β and Retinoic Acid-Activated Regulator of Cell Death

A combination of interferon-beta (IFN-beta) and all-trans retinoic acid (IFN/RA) induces tumor cell apoptosis via some unknown mechanisms. Apoptosis is a gene-directed process that limits the proliferation of undesired cells. Several genes are required to regulate cell death in the higher-order animals. Earlier, we employed a gene expression knockout technique to isolate cell death-related genes. A novel gene, the gene associated with retinoid-interferon-induced mortality-19 (GRIM-19), was found to be essential for tumor cell death induced by IFN/RA. Here, we describe the development and characterization of three monoclonal antibodies (mAbs) against GRIM-19. GRIM-19 is present in the nucleus and cytoplasm. Its expression is induced by the IFN/RA combination. We also show that GRIM-19 inhibits the cell-transforming property of viral oncogenic protein viral IFN regulatory factor-1 (vIRF-1) via a physical interaction. mAbs developed in this study should be useful for studying the other physiologic roles of GRIM-19 and serve as a potent tool for studying tumor responses to IFN/RA therapy.

IRF-2 regulates NF-κB activity by modulating the subcellular localization of NF-κB

Nuclear Factor-kappa B (NF-kappaB) is a transcription factor essential to the control of cell proliferation, survival, differentiation, immune response, and inflammation. Constitutive NF-kappaB activation has been observed in a broad variety of solid tumors and hematological malignancies, which suggests that NF-kappaB signaling may perform a critical role in the development of human cancers. Interferon regulatory factor-2 (IRF-2), an antagonistic transcriptional repressor of IRF-1, evidences oncogenic potential, but little is currently known regarding the mechanism underlying the oncogenic activities of IRF-2. In this study, we report that IRF-2 recruits RelA/p65 transcription factors into the nucleus via physical interaction. While the nuclear recruitment of RelA by IRF-2 augments TNFalpha-induced NF-kappaB dependent transcription, the N-terminal truncated mutant form of IRF-2 inhibits the nuclear localization of RelA, and thus interferes with NF-kappaB activation. Furthermore, the knockdown of IRF-2 by IRF-2 siRNA attenuates TNFalpha-induced NF-kappaB dependent transcription by inhibiting the nuclear localization of RelA. Thus, these results show that IRF-2 regulates NF-kappaB activity via the modulation of NF-kappaB subcellular localization.

The viral oncogene human papillomavirus E7 deregulates transcriptional silencing by Brm-related gene 1 via molecular interactions.

BRG-1, a component of the human SWI/SNF complex, either activates or represses cellular promoters by modulating chromatin structure via the formation of a multiple polypeptide complex. Human papillomavirus E7 binds and destabilizes pRb, resulting in the blockage of G1 arrest in the cell cycle. We show here that the high-risk human papillomavirus E7 protein group binds BRG-1 and modulates repression of the c-fospromoter mediated by this protein. In addition, both wild-type and Rb binding-defective E7 proteins abolish flat cell formation by BRG-1 in SW13 cells, whereas E7 COOH-terminal mutants do not affect this process. BRG-1-triggered repression of the c-fos promoter is sensitive to trichostatin A. We further establish that BRG-1 contains an activation domain and a trichostatin A-sensitive repression domain. These results collectively suggest that the viral oncogene E7 targets both pRb and BRG-1 via protein-protein interactions, resulting in the deregulation of host cell cycle control.