scholarly journals NEDDylation Is Essential for Kaposi’s Sarcoma-Associated Herpesvirus Latency and Lytic Reactivation and Represents a Novel Anti-KSHV Target

2015 ◽  
Vol 11 (3) ◽  
pp. e1004771 ◽  
Author(s):  
David J. Hughes ◽  
Jennifer J. Wood ◽  
Brian R. Jackson ◽  
Belinda Baquero-Pérez ◽  
Adrian Whitehouse
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Reactivation ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

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.

scholarly journals Principal Role of TRAP/Mediator and SWI/SNF Complexes in Kaposi's Sarcoma-Associated Herpesvirus RTA-Mediated Lytic Reactivation

2003 ◽  
Vol 23 (6) ◽  
pp. 2055-2067 ◽  
Author(s):  
Yousang Gwack ◽  
Hwa Jin Baek ◽  
Hiroyuki Nakamura ◽  
Sun Hwa Lee ◽  
Michael Meisterernst ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Viral Gene Expression ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Viral Gene ◽  
Transcription Activator ◽  
Lytic Reactivation ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT An important step in the herpesvirus life cycle is the switch from latency to lytic reactivation. The RTA transcription activator of Kaposi's sarcoma-associated herpesvirus (KSHV) acts as a molecular switch for lytic reactivation. Here we demonstrate that KSHV RTA recruits CBP, the SWI/SNF chromatin remodeling complex, and the TRAP/Mediator coactivator into viral promoters through interactions with a short acidic sequence in the carboxyl region and that this recruitment is essential for RTA-dependent viral gene expression. The Brg1 subunit of SWI/SNF and the TRAP230 subunit of TRAP/Mediator were shown to interact directly with RTA. Consequently, genetic ablation of these interactions abolished KSHV lytic replication. These results demonstrate that the recruitment of CBP, SWI/SNF, and TRAP/Mediator complexes by RTA is the principal mechanism to direct well-controlled viral gene expression and thereby viral lytic reactivation.

scholarly journals Expression and Subcellular Localization of the Kaposi's Sarcoma-Associated Herpesvirus K15P Protein during Latency and Lytic Reactivation in Primary Effusion Lymphoma Cells

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Caitlin G. Smith ◽  
Himanshu Kharkwal ◽  
Duncan W. Wilson
Keyword(s):  
Molecular Weight ◽  
Kaposi's Sarcoma ◽  
Primary Effusion Lymphoma ◽  
Kaposi’S Sarcoma ◽  
Full Length ◽  
Transcription Activator ◽  
Lytic Reactivation ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Different Molecular Weight

ABSTRACT The K15P membrane protein of Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with multiple cellular signaling pathways and is thought to play key roles in KSHV-associated endothelial cell angiogenesis, regulation of B-cell receptor (BCR) signaling, and the survival, activation, and proliferation of BCR-negative primary effusion lymphoma (PEL) cells. Although full-length K15P is ∼45 kDa, numerous lower-molecular-weight forms of the protein exist as a result of differential splicing and poorly characterized posttranslational processing. K15P has been reported to localize to numerous subcellular organelles in heterologous expression studies, but there are limited data concerning the sorting of K15P in KSHV-infected cells. The relationships between the various molecular weight forms of K15P, their subcellular distribution, and how these may differ in latent and lytic KSHV infections are poorly understood. Here we report that a cDNA encoding a full-length, ∼45-kDa K15P reporter protein is expressed as an ∼23- to 24-kDa species that colocalizes with the trans-Golgi network (TGN) marker TGN46 in KSHV-infected PEL cells. Following lytic reactivation by sodium butyrate, the levels of the ∼23- to 24-kDa protein diminish, and the full-length, ∼45-kDa K15P protein accumulates. This is accompanied by apparent fragmentation of the TGN and redistribution of K15P to a dispersed peripheral location. Similar results were seen when lytic reactivation was stimulated by the KSHV protein replication and transcription activator (RTA) and during spontaneous reactivation. We speculate that expression of different molecular weight forms of K15P in distinct cellular locations reflects the alternative demands placed upon the protein in the latent and lytic phases. IMPORTANCE The K15P protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is thought to play key roles in disease, including KSHV-associated angiogenesis and the survival and growth of primary effusion lymphoma (PEL) cells. The protein exists in multiple molecular weight forms, and its intracellular trafficking is poorly understood. Here we demonstrate that the molecular weight form of a reporter K15P molecule and its intracellular distribution change when KSHV switches from its latent (quiescent) phase to the lytic, infectious state. We speculate that expression of different molecular weight forms of K15P in distinct cellular locations reflects the alternative demands placed upon the protein in the viral latent and lytic stages.

scholarly journals Nuclear Innate Immune DNA Sensor IFI16 Is Degraded during Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus (KSHV): Role of IFI16 in Maintenance of KSHV Latency

2016 ◽  
Vol 90 (19) ◽  
pp. 8822-8841 ◽  
Author(s):  
Arunava Roy ◽  
Dipanjan Dutta ◽  
Jawed Iqbal ◽  
Gina Pisano ◽  
Olsi Gjyshi ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Innate Immune ◽  
Lytic Cycle ◽  
Lytic Gene ◽  
Lytic Reactivation ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTIFI16 (interferon gamma-inducible protein 16) recognizes nuclear episomal herpesvirus (Kaposi's sarcoma-associated herpesvirus [KSHV], Epstein-Barr virus [EBV], and herpes simplex virus 1 [HSV-1]) genomes and induces the inflammasome and interferon beta responses. It also acts as a lytic replication restriction factor and inhibits viral DNA replication (human cytomegalovirus [HCMV] and human papillomavirus [HPV]) and transcription (HSV-1, HCMV, and HPV) through epigenetic modifications of the viral genomes. To date, the role of IFI16 in the biology of latent viruses is not known. Here, we demonstrate that knockdown of IFI16 in the latently KSHV-infected B-lymphoma BCBL-1 and BC-3 cell lines results in lytic reactivation and increases in levels of KSHV lytic transcripts, proteins, and viral genome replication. Similar results were also observed during KSHV lytic cycle induction in TREX-BCBL-1 cells with the doxycycline-inducible lytic cycle switch replication and transcription activator (RTA) gene. Overexpression of IFI16 reduced lytic gene induction by the chemical agent 12-O-tetradecoylphorbol-13-acetate (TPA). IFI16 protein levels were significantly reduced or absent in TPA- or doxycycline-induced cells expressing lytic KSHV proteins. IFI16 is polyubiquitinated and degraded via the proteasomal pathway. The degradation of IFI16 was absent in phosphonoacetic acid-treated cells, which blocks KSHV DNA replication and, consequently, late lytic gene expression. Chromatin immunoprecipitation assays of BCBL-1 and BC-3 cells demonstrated that IFI16 binds to KSHV gene promoters. Uninfected epithelial SLK and osteosarcoma U2OS cells transfected with KSHV luciferase promoter constructs confirmed that IFI16 functions as a transcriptional repressor. These results reveal that KSHV utilizes the innate immune nuclear DNA sensor IFI16 to maintain its latency and repression of lytic transcripts, and a late lytic KSHV gene product(s) targets IFI16 for degradation during lytic reactivation.IMPORTANCELike all herpesviruses, latency is an integral part of the life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent for many human cancers. Herpesviruses utilize viral and host factors to successfully evade the host immune system to maintain latency. Reactivation is a complex event where the latent episomal viral genome springs back to active transcription of lytic cycle genes. Our studies reveal that KSHV has evolved to utilize the innate immune sensor IFI16 to keep lytic cycle transcription in dormancy. We demonstrate that IFI16 binds to the lytic gene promoter, acts as a transcriptional repressor, and thereby helps to maintain latency. We also discovered that during the late stage of lytic replication, KSHV selectively degrades IFI16, thus relieving transcriptional repression. This is the first report to demonstrate the role of IFI16 in latency maintenance of a herpesvirus, and further understanding will lead to the development of strategies to eliminate latent infection.

scholarly journals Lytic Reactivation of the Kaposi’s sarcoma-associated herpesvirus (KSHV) is Accompanied by Major Nucleolar Alterations

2020 ◽  
Author(s):  
Nofar Atari ◽  
K. Shanmugha Rajan ◽  
Vaibhav Chikne ◽  
Smadar Cohen-Chalamish ◽  
Odelia Orbaum ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Nuclear Bodies ◽  
Productive Infection ◽  
Pml Nuclear Bodies ◽  
Nucleolar Proteins ◽  
Rrna Transcription ◽  
Lytic Reactivation ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

AbstractThe nucleolus is a sub-nuclear compartment whose primary function is the biogenesis of ribosomal subunits. Certain viral infections affect the morphology and composition of the nucleolar compartment and influence ribosomal RNA (rRNA) transcription and maturation. However, no description of nucleolar morphology and function during infection with Kaposi’s sarcoma-associated herpesvirus (KSHV) is available to date. Using immunofluorescence microscopy, we documented extensive destruction of the nuclear and nucleolar architecture during lytic reactivation of KSHV. This was manifested by redistribution of key nucleolar proteins, including the rRNA transcription factor, UBF, the essential pre-rRNA processing factor Fibrillarin, and the nucleolar multifunctional phosphoproteins Nucleophosmin (NPM1) and Nucleolin. Distinct delocalization patterns were evident; certain nucleolar proteins remained together whereas others dissociated, implying that nucleolar proteins undergo nonrandom programmed dispersion. Of note, neither Fibrillarin nor UBF colocalized with promyelocytic leukemia (PML) nuclear bodies or with the viral protein LANA-1, and their redistribution was not dependent on viral DNA replication or late viral gene expression. No significant changes in pre-rRNA levels and no accumulation of pre-rRNA intermediates were found by RT-qPCR and Northern blot analysis, respectively. Furthermore, fluorescent in situ hybridization (FISH), combined with immunofluorescence, revealed an overlap between Fibrillarin and internal transcribed spacer 1 (ITS1), which represents the primary product of the pre-rRNA, suggesting that the processing of rRNA proceeds during lytic reactivation. Finally, small changes in the levels of pseudouridylation were documented across the rRNA. Taken together, our results suggest that rRNA transcription and processing persist during lytic reactivation of KSHV, yet they may become uncoupled. Whether the observed nucleolar alterations favor productive infection or signify cellular anti-viral responses remains to be determined.Author SummaryWe describe the extensive destruction of the nuclear and nucleolar architecture during lytic reactivation of KSHV. Distinct delocalization patterns are illustrated: certain nucleolar proteins remained associated with each other whereas others dissociated, implying that nucleolar proteins undergo nonrandom programmed dispersion. Of note, no significant changes in pre-rRNA levels and no accumulation of pre-rRNA intermediates were found, suggesting that pre-RNA transcription and processing continue and could be uncoupled during lytic reactivation. Small changes in the levels of pseudouridylation were documented across the rRNA. Previous studies showed that the different forms of KSHV infection are controlled through cellular and viral functions, which reprogram host epigenetic, transcriptomic, post-transcriptomic and proteomic landscapes. The ability of KSHV to affect the nucleolus and rRNA modifications constitutes a novel interaction network between viral and cellular components. The study of rRNA modifications is still in its infancy; however, the notion of altering cell fate by regulating rRNA modifications has recently begun to emerge, and its significance in viral infection is intriguing.

scholarly journals ARID3B: a Novel Regulator of the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle

2016 ◽  
Vol 90 (20) ◽  
pp. 9543-9555 ◽  
Author(s):  
Jennifer J. Wood ◽  
James R. Boyne ◽  
Christina Paulus ◽  
Brian R. Jackson ◽  
Michael M. Nevels ◽  
...  
Keyword(s):  
Gene Expression ◽  
Disease Progression ◽  
Causative Agent ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Cellular Protein ◽  
Lytic Cycle ◽  
Lytic Reactivation ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of commonly fatal malignancies of immunocompromised individuals, including primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS). A hallmark of all herpesviruses is their biphasic life cycle—viral latency and the productive lytic cycle—and it is well established that reactivation of the KSHV lytic cycle is associated with KS pathogenesis. Therefore, a thorough appreciation of the mechanisms that govern reactivation is required to better understand disease progression. The viral protein replication and transcription activator (RTA) is the KSHV lytic switch protein due to its ability to drive the expression of various lytic genes, leading to reactivation of the entire lytic cycle. While the mechanisms for activating lytic gene expression have received much attention, how RTA impacts cellular function is less well understood. To address this, we developed a cell line with doxycycline-inducible RTA expression and applied stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics. Using this methodology, we have identified a novel cellular protein (AT-rich interacting domain containing 3B [ARID3B]) whose expression was enhanced by RTA and that relocalized to replication compartments upon lytic reactivation. We also show that small interfering RNA (siRNA) knockdown or overexpression of ARID3B led to an enhancement or inhibition of lytic reactivation, respectively. Furthermore, DNA affinity and chromatin immunoprecipitation assays demonstrated that ARID3B specifically interacts with A/T-rich elements in the KSHV origin of lytic replication (oriLyt), and this was dependent on lytic cycle reactivation. Therefore, we have identified a novel cellular protein whose expression is enhanced by KSHV RTA with the ability to inhibit KSHV reactivation.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of fatal malignancies of immunocompromised individuals, including Kaposi's sarcoma (KS). Herpesviruses are able to establish a latent infection, in which they escape immune detection by restricting viral gene expression. Importantly, however, reactivation of productive viral replication (the lytic cycle) is necessary for the pathogenesis of KS. Therefore, it is important that we comprehensively understand the mechanisms that govern lytic reactivation, to better understand disease progression. In this study, we have identified a novel cellular protein (AT-rich interacting domain protein 3B [ARID3B]) that we show is able to temper lytic reactivation. We showed that the master lytic switch protein, RTA, enhanced ARID3B levels, which then interacted with viral DNA in a lytic cycle-dependent manner. Therefore, we have added a new factor to the list of cellular proteins that regulate the KSHV lytic cycle, which has implications for our understanding of KSHV biology.

scholarly journals Increased Efficiency of Phorbol Ester-Induced Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus during S Phase

2005 ◽  
Vol 79 (4) ◽  
pp. 2626-2630 ◽  
Author(s):  
Shane C. McAllister ◽  
Scott G. Hansen ◽  
Ilhem Messaoudi ◽  
Janko Nikolich-Zugich ◽  
Ashlee V. Moses
Keyword(s):  
Phorbol Ester ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
S Phase ◽  
Progeny Virus ◽  
Lytic Reactivation ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Lytic Genes

ABSTRACT Expression of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic genes is thought to be essential for the establishment and progression of KSHV-induced diseases. The inefficiency of lytic reactivation in various in vitro systems hampers the study of lytic genes in the context of whole virus. We report here increased expression of KSHV lytic genes and increased release of progeny virus when synchronized cultures of body cavity-based lymphoma-1 cells are treated with a phorbol ester during S phase of the cell cycle.

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.

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