scholarly journals Identification of Direct Transcriptional Targets of the Kaposi's Sarcoma-Associated Herpesvirus Rta Lytic Switch Protein by Conditional Nuclear Localization

2008 ◽  
Vol 82 (21) ◽  
pp. 10709-10723 ◽  
Author(s):  
Wei Bu ◽  
Diana Palmeri ◽  
Raghu Krishnan ◽  
Roxana Marin ◽  
Virginie M. Aris ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Nuclear Localization ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Cycle ◽  
Viral Reactivation ◽  
Cell Nuclei ◽  
Transcriptional Targets ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Lytic reactivation from latency is critical for the pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV). We previously demonstrated that the 691-amino-acid (aa) KSHV Rta transcriptional transactivator is necessary and sufficient to reactivate the virus from latency. Viral lytic cycle genes, including those expressing additional transactivators and putative oncogenes, are induced in a cascade fashion following Rta expression. In this study, we sought to define Rta's direct targets during reactivation by generating a conditionally nuclear variant of Rta. Wild-type Rta protein is constitutively localized to cell nuclei and contains two putative nuclear localization signals (NLSs). Only one NLS (NLS2; aa 516 to 530) was required for the nuclear localization of Rta, and it relocalized enhanced green fluorescent protein exclusively to cell nuclei. The results of analyses of Rta NLS mutants demonstrated that proper nuclear localization of Rta was required for transactivation and the stimulation of viral reactivation. RTA with NLS1 and NLS2 deleted was fused to the hormone-binding domain of the murine estrogen receptor to generate an Rta variant whose nuclear localization and ability to transactivate and induce reactivation were tightly controlled posttranslationally by the synthetic hormone tamoxifen. We used this strategy in KSHV-infected cells treated with protein synthesis inhibitors to identify direct transcriptional targets of Rta. Rta activated only eight KSHV genes in the absence of de novo protein synthesis. These direct transcriptional targets of Rta were transactivated to different levels and included the genes nut-1/PAN, ORF57/Mta, ORF56/Primase, K2/viral interleukin-6 (vIL-6), ORF37/SOX, K14/vOX, K9/vIRF1, and ORF52. Our data suggest that the induction of most of the KSHV lytic cycle genes requires additional protein expression after the expression of Rta.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Lytic Switch Protein Stimulates DNA Binding of RBP-Jk/CSL To Activate the Notch Pathway

2006 ◽  
Vol 80 (19) ◽  
pp. 9697-9709 ◽  
Author(s):  
Kyla Driscoll Carroll ◽  
Wei Bu ◽  
Diana Palmeri ◽  
Sophia Spadavecchia ◽  
Stephen J. Lynch ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Kaposi's Sarcoma ◽  
Notch Pathway ◽  
Kaposi’S Sarcoma ◽  
Lytic Cycle ◽  
Viral Reactivation ◽  
Transactivation Domain ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) lytic switch protein, Rta, is a ligand-independent inducer of the Notch signal transduction pathway, and KSHV cannot reactivate from latency in cells null for the Notch target protein RBP-Jk. Here we show that Rta promotes DNA binding of RBP-Jk, a mechanism that is fundamentally different from that established for the RBP-Jk-activating proteins, Notch intracellular domain (NICD) and Epstein-Barr virus EBNA2. Although constitutively active RBP-Jk and NICD do not transactivate KSHV promoters independently, cotransfection of an Rta mutant lacking its transactivation domain robustly restores transcriptional activation. Cooperation requires intact DNA binding sites for Rta and RBP-Jk and trimeric complex formation between the three molecules in vitro. In infected cells, RBP-Jk is virtually undetectable on a series of viral and cellular promoters during KSHV latency but is significantly enriched following Rta expression during viral reactivation. Accordingly, Rta, but not EBNA2 and NICD, reactivates the complete viral lytic cycle.

scholarly journals Kaposi’s Sarcoma-Associated Herpesvirus Lytic Replication Interferes with mTORC1 Regulation of Autophagy and Viral Protein Synthesis

2019 ◽  
Vol 93 (21) ◽  
Author(s):  
Eric S. Pringle ◽  
Carolyn-Ann Robinson ◽  
Craig McCormick
Keyword(s):  
Protein Synthesis ◽  
Viral Protein ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Viral Protein Synthesis ◽  
Global Protein Synthesis ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cellular metabolism. In nutrient-rich environments, mTORC1 kinase activity stimulates protein synthesis to meet cellular anabolic demands. Under nutrient-poor conditions or under stress, mTORC1 is rapidly inhibited, global protein synthesis is arrested, and a cellular catabolic process known as autophagy is activated. Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes multiple proteins that stimulate mTORC1 activity or subvert autophagy, but precise roles for mTORC1 in different stages of KSHV infection remain incompletely understood. Here, we report that during latent and lytic stages of KSHV infection, chemical inhibition of mTORC1 caused eukaryotic initiation factor 4F (eIF4F) disassembly and diminished global protein synthesis, which indicated that mTORC1-mediated control of translation initiation was largely intact. We observed that mTORC1 was required for synthesis of the replication and transcription activator (RTA) lytic switch protein and reactivation from latency, but once early lytic gene expression had begun, mTORC1 was not required for genome replication, late gene expression, or the release of infectious progeny. Moreover, mTORC1 control of autophagy was dysregulated during lytic replication, whereby chemical inhibition of mTORC1 prevented ULK1 phosphorylation but did not affect autophagosome formation or rates of autophagic flux. Together, these findings suggest that mTORC1 is dispensable for viral protein synthesis and viral control of autophagy during lytic infection and that KSHV undermines mTORC1-dependent cellular processes during the lytic cycle to ensure efficient viral replication. IMPORTANCE All viruses require host cell machinery to synthesize viral proteins. A host cell protein complex known as mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of protein synthesis. Under nutrient-rich conditions, mTORC1 is active and promotes protein synthesis to meet cellular anabolic demands. Under nutrient-poor conditions or under stress, mTORC1 is rapidly inhibited, global protein synthesis is arrested, and a cellular catabolic process known as autophagy is activated. Kaposi’s sarcoma-associated herpesvirus (KSHV) stimulates mTORC1 activity and utilizes host machinery to synthesize viral proteins. However, we discovered that mTORC1 activity was largely dispensable for viral protein synthesis, genome replication, and the release of infectious progeny. Likewise, during lytic replication, mTORC1 was no longer able to control autophagy. These findings suggest that KSHV undermines mTORC1-dependent cellular processes during the lytic cycle to ensure efficient viral replication.

scholarly journals Effects of the NEDD8-Activating Enzyme Inhibitor MLN4924 on Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus

2017 ◽  
Vol 91 (19) ◽  
Author(s):  
Pey-Jium Chang ◽  
Lee-Wen Chen ◽  
Li-Yu Chen ◽  
Chien-Hui Hung ◽  
Ying-Ju Shih ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Cell Lines ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Viral Reactivation ◽  
Lytic Reactivation ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The switch of Kaposi's sarcoma-associated herpesvirus (KSHV) from latency to lytic replication is a key event for viral dissemination and pathogenesis. MLN4924, a novel neddylation inhibitor, reportedly causes the onset of KSHV reactivation but impairs later phases of the viral lytic program in infected cells. Thus far, the molecular mechanism involved in the modulation of the KSHV lytic cycle by MLN4924 is not yet fully understood. Here, we confirmed that treatment of different KSHV-infected primary effusion lymphoma (PEL) cell lines with MLN4924 substantially induces viral lytic protein expression. Due to the key role of the virally encoded ORF50 protein in the latent-to-lytic switch, we investigated its transcriptional regulation by MLN4924. We found that MLN4924 activates the ORF50 promoter (ORF50p) in KSHV-positive cells (but not in KSHV-negative cells), and the RBP-Jκ-binding elements within the promoter are critically required for MLN4924 responsiveness. In KSHV-negative cells, reactivation of the ORF50 promoter by MLN4924 requires the presence of the latency-associated nuclear antigen (LANA). Under such a condition, LANA acts as a repressor to block the ORF50p activity, whereas MLN4924 treatment relieves LANA-mediated repression. Importantly, we showed that LANA is a neddylated protein and can be deneddylated by MLN4924. On the other hand, we revealed that MLN4924 exhibits concentration-dependent biphasic effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)- or sodium butyrate (SB)-induced viral reactivation in PEL cell lines. In other words, low concentrations of MLN4924 promote activation of TPA- or SB-mediated viral reactivation, whereas high concentrations of MLN4924, conversely, inhibit the progression of TPA- or SB-mediated viral lytic program. IMPORTANCE MLN4924 is a neddylation (NEDD8 modification) inhibitor, which currently acts as an anti-cancer drug in clinical trials. Although MLN4924 has been reported to trigger KSHV reactivation, many aspects regarding the action of MLN4924 in regulating the KSHV lytic cycle are not fully understood. Since the KSHV ORF50 protein is the key regulator of viral lytic reactivation, we focus on its transcriptional regulation by MLN4924. We here show that activation of the ORF50 gene by MLN4924 involves the relief of LANA-mediated transcriptional repression. Importantly, we find that LANA is a neddylated protein. To our knowledge, this is the first report showing that neddylation occurs in viral proteins. Additionally, we provide evidence that different concentrations of MLN4924 have opposite effects on TPA-mediated or SB-mediated KSHV lytic cycle activation. Therefore, in clinical application, we propose that MLN4924 needs to be used with caution in combination therapy to treat KSHV-positive subjects.

scholarly journals Identification of the Immediate-Early Transcripts of Kaposi’s Sarcoma-Associated Herpesvirus

1999 ◽  
Vol 73 (7) ◽  
pp. 5556-5567 ◽  
Author(s):  
Fan Xiu Zhu ◽  
Teresa Cusano ◽  
Yan Yuan
Keyword(s):  
Protein Synthesis ◽  
Kaposi's Sarcoma ◽  
Screening Method ◽  
Kaposi’S Sarcoma ◽  
Viral Reactivation ◽  
Immediate Early ◽  
Transcription Activator ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Inhibitor Of Protein Synthesis ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT In the immediate-early phase of reactivation or primary infection, herpesviruses express a small number of genes without requiring prior viral protein synthesis. Immediate-early genes usually encode regulatory proteins critical for the viral life cycle. Kaposi’s sarcoma-associated herpesvirus (KSHV) gene transcription in the immediate-early stage of viral reactivation was examined by using a chemical induction combined with a gene expression screening method. RNA transcripts from at least four KSHV genomic loci accumulate when latently infected B-lymphoma cells are induced for reactivation in the presence of an inhibitor of protein synthesis (cycloheximide) and thus represent immediate-early class transcripts. Among them, a 3.6-kb mRNA encodes three putative open reading frames (ORFs), namely, ORF50, K8, and K8.2. ORF50 is a homologue of Rta, a transcription activator encoded by Epstein-Barr virus (EBV). The K8 gene codes for a 237-amino-acid protein with a basic-leucine zipper domain near its C terminus and an acidic domain near its N terminus and which closely resembles the ZEBRA protein of EBV and Jun/Fos family proteins. Other immediate-early mRNAs of KSHV include a 1.7-kb mRNA encoding ORF45, a 2.0-kb mRNA encoding ORF K4.2, and a 4.5-kb mRNA. Functional roles of products of these KSHV immediate-early transcripts remain to be studied.

scholarly journals Full-Length Isoforms of Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Accumulate in the Cytoplasm of Cells Undergoing the Lytic Cycle of Replication

2017 ◽  
Vol 91 (24) ◽  
Author(s):  
H. Jacques Garrigues ◽  
Kellie Howard ◽  
Serge Barcy ◽  
Minako Ikoma ◽  
Ashlee V. Moses ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Localization ◽  
Primary Infection ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Nuclear Antigen ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) performs a variety of functions to establish and maintain KSHV latency. During latency, LANA localizes to discrete punctate spots in the nucleus, where it tethers viral episomes to cellular chromatin and interacts with nuclear components to regulate cellular and viral gene expression. Using highly sensitive tyramide signal amplification, we determined that LANA localizes to the cytoplasm in different cell types undergoing the lytic cycle of replication after de novo primary infection and after spontaneous, tetradecanoyl phorbol acetate-, or open reading frame 50 (ORF50)/replication transactivator (RTA)-induced activation. We confirmed the presence of cytoplasmic LANA in a subset of cells in lytically active multicentric Castleman disease lesions. The induction of cellular migration by scratch-wounding confluent cell cultures, culturing under subconfluent conditions, or induction of cell differentiation in primary cultures upregulated the number of cells permissive for primary lytic KSHV infection. The induction of lytic replication was characterized by high-level expression of cytoplasmic LANA and nuclear ORF59, a marker of lytic replication. Subcellular fractionation studies revealed the presence of multiple isoforms of LANA in the cytoplasm of ORF50/RTA-activated Vero cells undergoing primary infection. Mass spectrometry analysis demonstrated that cytoplasmic LANA isoforms were full length, containing the N-terminal nuclear localization signal. These results suggest that trafficking of LANA to different subcellular locations is a regulated phenomenon, which allows LANA to interact with cellular components in different compartments during both the latent and the replicative stages of the KSHV life cycle. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) causes AIDS-related malignancies, including lymphomas and Kaposi's sarcoma. KSHV establishes lifelong infections using its latency-associated nuclear antigen (LANA). During latency, LANA localizes to the nucleus, where it connects viral and cellular DNA complexes and regulates gene expression, allowing the virus to maintain long-term infections. Our research shows that intact LANA traffics to the cytoplasm of cells undergoing permissive lytic infections and latently infected cells in which the virus is induced to replicate. This suggests that LANA plays important roles in the cytoplasm and nuclear compartments of the cell during different stages of the KSHV life cycle. Determining cytoplasmic function and mechanism for regulation of the nuclear localization of LANA will enhance our understanding of the biology of this virus, leading to therapeutic approaches to eliminate infection and block its pathological effects.

scholarly journals Activation of the Unfolded Protein Response by 2-Deoxy-d-Glucose Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication and Gene Expression

2012 ◽  
Vol 56 (11) ◽  
pp. 5794-5803 ◽  
Author(s):  
Howard J. Leung ◽  
Elda M. Duran ◽  
Metin Kurtoglu ◽  
Samita Andreansky ◽  
Theodore J. Lampidis ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Unfolded Protein ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Protein Response ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTLytic replication of the Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for the maintenance of both the infected state and characteristic angiogenic phenotype of Kaposi's sarcoma and thus represents a desirable therapeutic target. During the peak of herpesvirus lytic replication, viral glycoproteins are mass produced in the endoplasmic reticulum (ER). Normally, this leads to ER stress which, through an unfolded protein response (UPR), triggers phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α), resulting in inhibition of protein synthesis to maintain ER and cellular homeostasis. However, in order to replicate, herpesviruses have acquired the ability to prevent eIF2α phosphorylation. Here we show that clinically achievable nontoxic doses of the glucose analog 2-deoxy-d-glucose (2-DG) stimulate ER stress, thereby shutting down eIF2α and inhibiting KSHV and murine herpesvirus 68 replication and KSHV reactivation from latency. Viral cascade genes that are involved in reactivation, including the master transactivator (RTA) gene, glycoprotein B, K8.1, and angiogenesis-regulating genes are markedly decreased with 2-DG treatment. Overall, our data suggest that activation of UPR by 2-DG elicits an early antiviral response via eIF2α inactivation, which impairs protein synthesis required to drive viral replication and oncogenesis. Thus, induction of ER stress by 2-DG provides a new antiherpesviral strategy that may be applicable to other viruses.

scholarly journals Identification of the Nuclear Export and Adjacent Nuclear Localization Signals for ORF45 of Kaposi's Sarcoma-Associated Herpesvirus

2008 ◽  
Vol 83 (6) ◽  
pp. 2531-2539 ◽  
Author(s):  
Xiaojuan Li ◽  
Fanxiu Zhu
Keyword(s):  
Subcellular Localization ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Kaposi's Sarcoma ◽  
Nuclear Export Signal ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Wild Type ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus 8 (KSHV) is an immediate-early phosphorylated tegument protein and has been shown to play important roles at both early and late stages of viral infection. Homologues of ORF45 exist only in gammaherpesviruses, and their homology is limited. These homologues differ in their protein lengths and subcellular localizations. We and others have reported that KSHV ORF45 is localized predominantly in the cytoplasm, whereas its homologue in murine herpesvirus 68 is localized exclusively in the nucleus. We observed that ORF45s of rhesus rhadinovirus and herpesvirus saimiri are found exclusively in the nucleus. As a first step toward understanding the mechanism underlying the distinct intracellular distribution of KSHV ORF45, we identified the signals that control its subcellular localization. We found that KSHV ORF45 accumulated rapidly in the nucleus in the presence of leptomycin B, an inhibitor of CRM1 (exportin 1)-dependent nuclear export, suggesting that it could shuttle between the nucleus and cytoplasm. Mutational analysis revealed that KSHV ORF45 contains a CRM1-dependent, leucine-rich-like nuclear export signal and an adjacent nuclear localization signal. Replacement of the key residues with alanines in these motifs of ORF45 disrupts its shuttling between the cytoplasm and nucleus. The resulting ORF45 mutants have restricted subcellular localizations, being found exclusively either in the cytoplasm or in the nucleus. Recombinant viruses were reconstituted by introduction of these mutations into KSHV bacterial artificial chromosome BAC36. The resultant viruses have distinct phenotypes. A mutant virus in which ORF45 is restricted to the cytoplasm behaves as an ORF45-null mutant and produces 5- to 10-fold fewer progeny viruses than the wild type. In contrast, mutants in which the ORF45 protein is mostly restricted to the nucleus produce numbers of progeny viruses similar to those produced by the wild type. These data suggest that the subcellular localization signals of ORF45 have important functional roles in KSHV lytic replication.

scholarly journals Suppression of Tetradecanoyl Phorbol Acetate-Induced Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus by K1 Signal Transduction

2002 ◽  
Vol 76 (23) ◽  
pp. 12185-12199 ◽  
Author(s):  
Bok-Soo Lee ◽  
Mini Paulose-Murphy ◽  
Young-Hwa Chung ◽  
Michelle Connlole ◽  
Steven Zeichner ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Kaposi's Sarcoma ◽  
Ectopic Expression ◽  
Kaposi’S Sarcoma ◽  
Viral Reactivation ◽  
Dependent Manner ◽  
Tetradecanoyl Phorbol Acetate ◽  
Lytic Reactivation ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic region and elicits cellular signal transduction through this motif. To investigate the role of K1 signal transduction in KSHV replication, we expressed full-length K1 and CD8-K1 chimeras in BCBL1 cells. Unlike its strong signaling activity in uninfected B lymphocytes, K1 did not induce intracellular calcium mobilization or NF-AT activation at detectable levels in KSHV-infected BCBL1 cells. Instead, K1 signaling dramatically suppressed KSHV lytic reactivation induced by tetradecanoyl phorbol acetate (TPA) stimulation, but not by ORF50 ectopic expression. Mutational analysis showed that the cytoplasmic ITAM sequence of K1 was required for this suppression. Viral microarray and immunoblot analyses demonstrated that K1 signaling suppressed the TPA-mediated increase in the expression of a large subset of viral lytic genes in KSHV-infected BCBL1 cells. Furthermore, electrophoretic mobility shift assays demonstrated that TPA-induced activation of AP-1, NF-κB, and Oct-1 activities was severely diminished in BCBL1 cells expressing the K1 cytoplasmic domain. The reduced activities of these transcription factors may confer the observed reduction in viral lytic gene expression. These results demonstrate that K1-mediated signal transduction in KSHV-infected cells is profoundly different from that in KSHV-negative cells. Furthermore, K1 signal transduction efficiently suppresses TPA-mediated viral reactivation in an ITAM-dependent manner, and this suppression may contribute to the establishment and/or maintenance of KSHV latency in vivo.

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