Amino acids essential for the interaction between cellular heat shock protein 90 and a Kaposi’s sarcoma-associated herpesvirus-encoded protein kinase ORF36

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus that has been implicated in the pathogenesis of Kaposi's sarcoma and B-cell neoplasms. The genomic organization of KSHV is similar to that of Epstein-Barr virus (EBV). EBV encodes two transcriptional factors, Rta and Zta, which functionally interact to transactivate EBV genes during replication and reactivation from latency. KSHV encodes a basic leucine zipper protein (K-bZIP), a homologue of EBV Zta, and K-Rta, the homologue of EBV Rta. EBV Rta and Zta are strong transcriptional transactivators. Although there is ample evidence that K-Rta is a potent transactivator, the role of K-bZIP as a transcriptional factor is much less clear. In this study, we report that K-bZIP modulates K-Rta function. We show that K-bZIP directly interacts with K-Rta in vivo and in vitro. This association is specific, requiring the basic domain (amino acids 122 to 189) of K-bZIP and a specific region (amino acids 499 to 550) of K-Rta, and can be detected with K-bZIP and K-Rta endogenously expressed in BCBL-1 cells treated with tetradecanoyl phorbol acetate. The functional relevance of this association was revealed by the observation that K-bZIP represses the transactivation of the ORF57 promoter by K-Rta in a dose-dependent manner. K-bZIP lacking the interaction domain fails to repress K-Rta-mediated transactivation; this finding attests to the specificity of the repression. Interestingly, this repression is not observed for the promoter of polyadenylated nuclear (PAN) RNA, another target of K-Rta; thus, repression is promoter dependent. Finally, we provide evidence that the modulation of K-Rta by K-bZIP also occurs in vivo during reactivation of the viral genome in BCBL-1 cells. When K-bZIP is overexpressed in BCBL-1 cells, the level of expression of ORF57 but not PAN RNA is repressed. These data support the model that one function of K-bZIP is to modulate the activity of the transcriptional transactivator K-Rta.

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NFAT and CREB Regulate Kaposi's Sarcoma-Associated Herpesvirus-Induced Cyclooxygenase 2 (COX-2)

Journal of Virology ◽

10.1128/jvi.01065-10 ◽

2010 ◽

Vol 84 (24) ◽

pp. 12733-12753 ◽

Cited By ~ 27

Author(s):

Neelam Sharma-Walia ◽

Arun George Paul ◽

Kinjan Patel ◽

Karthic Chandran ◽

Waseem Ahmad ◽

...

Keyword(s):

Transcriptional Regulation ◽

Protein Kinase ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Prostaglandin E ◽

Intracellular Camp ◽

Kshv Infection ◽

Cox 2 ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT COX-2 has been implicated in Kaposi's sarcoma-associated herpesvirus (KSHV) latency and pathogenesis (A. George Paul, N. Sharma-Walia, N. Kerur, C. White, and B. Chandran, Cancer Res. 70:3697-3708, 2010; P. P. Naranatt, H. H. Krishnan, S. R. Svojanovsky, C. Bloomer, S. Mathur, and B. Chandran, Cancer Res. 64:72-84, 2004; N. Sharma-Walia, A. G. Paul, V. Bottero, S. Sadagopan, M. V. Veettil, N. Kerur, and B. Chandran, PLoS Pathog. 6:e1000777, 2010; N. Sharma-Walia, H. Raghu, S. Sadagopan, R. Sivakumar, M. V. Veettil, P. P. Naranatt, M. M. Smith, and B. Chandran, J. Virol. 80:6534-6552, 2006). However, the precise regulatory mechanisms involved in COX-2 induction during KSHV infection have never been explored. Here, we identified cis-acting elements involved in the transcriptional regulation of COX-2 upon KSHV de novo infection. Promoter analysis using human COX-2 promoter deletion and mutation reporter constructs revealed that nuclear factor of activated T cells (NFAT) and the cyclic AMP (cAMP) response element (CRE) modulate KSHV-mediated transcriptional regulation of COX-2. Along with multiple KSHV-induced signaling pathways, infection-induced prostaglandin E2 (PGE2) also augmented COX-2 transcription. Infection of endothelial cells markedly induced COX-2 expression via a cyclosporine A-sensitive, calcineurin/NFAT-dependent pathway. KSHV infection increased intracellular cAMP levels and activated protein kinase A (PKA), which phosphorylated the CRE-binding protein (CREB) at serine 133, which probably led to interaction with CRE in the COX-2 promoter, thereby enhancing COX-2 transcription. PKA selective inhibitor H-89 pretreatment strongly inhibited CREB serine 133, indicating the involvement of a cAMP-PKA-CREB-CRE loop in COX-2 transcriptional regulation. In contrast to phosphatidylinositol 3-kinase and protein kinase C, inhibition of FAK and Src effectively reduced KSHV infection-induced COX-2 transcription and protein levels. Collectively, our study indicates that mediation of COX-2 transcription upon KSHV infection is a paradigm of a complex regulatory milieu involving the interplay of multiple signal cascades and transcription factors. Intervention at each step of COX-2/PGE2 induction can be used as a potential therapeutic target to treat KSHV-associated neoplasm and control inflammatory sequels of KSHV infection.

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A Domain in the C-Terminal Region of Latency-Associated Nuclear Antigen 1 of Kaposi's Sarcoma-Associated Herpesvirus Affects Transcriptional Activation and Binding to Nuclear Heterochromatin

Journal of Virology ◽

10.1128/jvi.77.12.7093-7100.2003 ◽

2003 ◽

Vol 77 (12) ◽

pp. 7093-7100 ◽

Cited By ~ 35

Author(s):

Abel Viejo-Borbolla ◽

Emrah Kati ◽

Julie A. Sheldon ◽

Kavita Nathan ◽

Karin Mattsson ◽

...

Keyword(s):

Amino Acids ◽

Nuclear Matrix ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Terminal Region ◽

Nuclear Antigen ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Carboxy Terminal ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Episomal Dna

ABSTRACT The latency-associated nuclear antigen 1 (LANA-1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for the maintenance and replication of viral episomal DNA. The binding sites for nuclear heterochromatin and transcriptional repressor complexes are located in an amino-terminal region of LANA-1, whereas those for viral episomal DNA, p53, pRB, and members of the BRD/fsh family of nuclear proteins are located in its carboxy-terminal domain. LANA-1 activates or represses several cellular and viral promoters. In this report we show that a domain of 15 amino acids (amino acids 1129 to 1143), located close to the carboxy-terminal end of LANA-1, is required for the interaction of LANA-1 with nuclear heterochromatin or nuclear matrix, and for the ability of LANA-1 to activate the Epstein-Barr virus Cp promoter. LANA-1 proteins that are tightly associated with nuclear heterochromatin or matrix differ in molecular weight from LANA-1 proteins that can be dissociated from the nuclear matrix by high-salt buffers, suggesting that posttranslational modifications may determine the association of LANA-1 with nuclear heterochromatin or matrix.

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Reactivation of Kaposi's sarcoma-associated herpesvirus from latency requires MEK/ERK, JNK and p38 multiple mitogen-activated protein kinase pathways

Virology ◽

10.1016/j.virol.2007.09.040 ◽

2008 ◽

Vol 371 (1) ◽

pp. 139-154 ◽

Cited By ~ 83

Author(s):

Jianping Xie ◽

Adetola Olalekan Ajibade ◽

Fengchun Ye ◽

Kurt Kuhne ◽

Shou-Jiang Gao

Keyword(s):

Protein Kinase ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Mitogen Activated Protein Kinase ◽

Mitogen Activated Protein ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

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The Cytoplasmic Terminus of Kaposi's Sarcoma-Associated Herpesvirus Glycoprotein B Is Not Essential for Virion Egress and Infectivity

Journal of Virology ◽

10.1128/jvi.00617-08 ◽

2008 ◽

Vol 82 (14) ◽

pp. 7144-7154 ◽

Cited By ~ 9

Author(s):

R. Subramanian ◽

O. D'Auvergne ◽

Haixia Kong ◽

K. G. Kousoulas

Keyword(s):

Amino Acids ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Cytoplasmic Domain ◽

Glycoprotein B ◽

Virus Infectivity ◽

Mrna Levels ◽

293 Cells ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded glycoprotein B (gB) is an important determinant of viral infectivity and virion egress. A small interfering RNA (siRNA)-based strategy was devised to inhibit KSHV gB gene expression. Transient cotransfection of plasmids constitutively expressing gB and anti-gB siRNAs in 293 cells substantially inhibited gB mRNA levels and protein production. Similarly, transient expression of siRNAs into the primary effusion lymphoma cell line BCBL-1 caused a substantial reduction of gB transcripts and protein synthesis. TaqMan real-time PCR assays against the lytic KSHV gene ORF59 and infectivity assays on 293 cells were employed to assess the effect of inhibiting gB synthesis on virion egress from BCBL-1 cells and infectivity on 293 cells, respectively. These experiments showed that gB was essential for virion egress and infectivity. Transfection of a codon-optimized gB gene with the first 540 nucleotides altered, and therefore not recognized by anti-gB siRNAs that target the native but not the codon-optimized sequence, efficiently rescued virion egress and infectivity in BCBL-1 cells in the presence of siRNAs inhibiting wild-type gB expression. To assess the role of the cytoplasmic domain of gB in virion egress, mutant gB genes were generated specifying carboxyl terminal truncations of 25 and 58 amino acids disrupting two prominent predicted α-helical domains associated with virus-induced cell fusion. A third truncation removed the entire predicted cytoplasmic terminus of 84 amino acids, while a fourth truncation removed 110 amino acids, including the terminal most hydrophobic, intramembrane anchoring sequence. Virion egress experiments revealed that all truncated gBs facilitated virion egress from BCBL-1 cells, with the exception of the largest 110-amino-acid truncation, which removed the gB anchoring sequence. Importantly, the gB truncation that removed the entire predicted cytoplasmic domain increased virion egress, suggesting the presence of a egress regulation domain located proximal to the intramembrane sequence within the cytoplasmic domain of gB. All supernatant virions were infectious on 293 cells, indicating that the carboxyl terminus of gB is not essential for either virion egress or virus infectivity.

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