X Box Binding Protein XBP-1s Transactivates the Kaposi's Sarcoma-Associated Herpesvirus (KSHV) ORF50 Promoter, Linking Plasma Cell Differentiation to KSHV Reactivation from Latency

ABSTRACT Reactivation of lytic replication from viral latency is a defining property of all herpesviruses. Despite this, the authentic physiological cues for the latent-lytic switch are unclear. Such cues should ensure that viral lytic replication occurs under physiological conditions, predominantly in sites which facilitate transmission to permissive uninfected cells and new susceptible hosts. Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with the B-cell neoplasm primary effusion lymphoma (PEL), in which the virus remains latent. We have previously shown that PEL cells have the gene expression profile and immunophenotype of cycling preplasma cells (plasmablasts). Here, we show that the highly active spliced isoform of plasma cell transcription factor X box binding protein 1 (XBP-1s) is a lytic switch for KSHV. XBP-1s is normally absent in PEL, but the induction of endoplasmic reticulum stress leads to XBP-1s generation, plasma cell-like differentiation, and lytic reactivation of KSHV. XBP-1s binds to and activates the KSHV immediate-early gene ORF50 and synergizes with the ORF50 gene product RTA to induce a full lytic cycle. These data suggest that KSHV remains latent until B-cell terminal differentiation into plasma cells, the transcriptional environment of which provides the physiological “lytic switch” through XBP-1s. This links B-cell terminal differentiation to KSHV lytic reactivation.

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Cellular Corepressor TLE2 Inhibits Replication-and-Transcription- Activator-Mediated Transactivation and Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus

Journal of Virology ◽

10.1128/jvi.01984-09 ◽

2009 ◽

Vol 84 (4) ◽

pp. 2047-2062 ◽

Cited By ~ 16

Author(s):

Zhiheng He ◽

Yunhua Liu ◽

Deguang Liang ◽

Zhuo Wang ◽

Erle S. Robertson ◽

...

Keyword(s):

Kaposi's Sarcoma ◽

Binding Protein ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Host Protein ◽

Transcription Activator ◽

Rich Domain ◽

Lytic Reactivation ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Replication and transcription activator (RTA) encoded by open reading frame 50 (ORF50) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential and sufficient to initiate lytic reactivation. RTA activates its target genes through direct binding with high affinity to its responsive elements or by interaction with cellular factors, such as RBP-Jκ, Ap-1, C/EBP-α, and Oct-1. In this study, we identified transducin-like enhancer of split 2 (TLE2) as a novel RTA binding protein by using yeast two-hybrid screening of a human spleen cDNA library. The interaction between TLE2 and RTA was confirmed by glutathione S-transferase (GST) binding and coimmunoprecipitation assays. Immunofluorescence analysis showed that TLE2 and RTA were colocalized in the same nuclear compartment in KSHV-infected cells. This interaction recruited TLE2 to RTA bound to its recognition sites on DNA and repressed RTA auto-activation and transactivation activity. Moreover, TLE2 also inhibited the induction of lytic replication and virion production driven by RTA. We further showed that the Q (Gln-rich), SP (Ser-Pro-rich), and WDR (Trp-Asp repeat) domains of TLE2 and the Pro-rich domain of RTA were essential for this interaction. RBP-Jκ has been shown previously to bind to the same Pro-rich domain of RTA, and this binding can be subject to competition by TLE2. In addition, TLE2 can form a complex with RTA to access the cognate DNA sequence of the RTA-responsive element at different promoters. Intriguingly, the transcription level of TLE2 could be upregulated by RTA during the lytic reactivation process. In conclusion, we identified a new RTA binding protein, TLE2, and demonstrated that TLE2 inhibited replication and transactivation mediated by RTA. This provides another potentially important mechanism for maintenance of KSHV viral latency through interaction with a host protein.

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

Molecular and Cellular Biology ◽

10.1128/mcb.23.6.2055-2067.2003 ◽

2003 ◽

Vol 23 (6) ◽

pp. 2055-2067 ◽

Cited By ~ 77

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.

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Induction of Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

Journal of Virology ◽

10.1128/jvi.01555-19 ◽

2019 ◽

Vol 94 (5) ◽

Author(s):

Victoria Wang ◽

David A. Davis ◽

Claire Deleage ◽

Catherine Brands ◽

Hong S. Choi ◽

...

Keyword(s):

B Cells ◽

Thymidine Kinase ◽

Germinal Center ◽

Kaposi's Sarcoma ◽

Binding Protein ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Germinal Center B Cells

ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). Like other herpesviruses, it has latent and lytic repertoires. However, there is evidence that some lytic genes can be directly activated by certain cellular factors. Cells undergoing endoplasmic reticulum stress express spliced X-box binding protein 1 (XBP-1s). XBP-1s is also present in large amounts in germinal center B cells. XBP-1s can activate the KSHV replication and transcription activator (RTA) and lytic replication. It can also directly activate KSHV-encoded viral interleukin-6 (vIL-6) and, thus, contribute to the pathogenesis of KSHV MCD. KSHV thymidine kinase (TK), the ORF21 gene product, can enhance the production of dTTP and is important for lytic replication. It can also phosphorylate zidovudine and ganciclovir to toxic moieties, enabling treatment of KSHV-MCD with these drugs. We show here that XBP-1s can directly activate ORF21 and that this activation is mediated primarily through two XBP-response elements (XRE) on the ORF21 promoter region. Deletion or mutation of these elements eliminated XBP-1s-induced upregulation of the promoter, and chromatin immunoprecipitation studies provide evidence that XBP-1s can bind to both XREs. Exposure of PEL cells to a chemical inducer of XBP-1s can induce ORF21 within 4 hours, and ORF21 expression in the lymph nodes of patients with KSHV-MCD is predominantly found in cells with XBP-1. Thus, XBP-1s may directly upregulate KSHV ORF21 and, thus, contribute to the pathogenesis of KSHV-MCD and the activity of zidovudine and valganciclovir in this disease. IMPORTANCE Spliced X-box binding protein 1 (XBP-1s), part of the unfolded protein response and expressed in developing germinal center B cells, can induce Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic replication and directly activate viral interleukin-6 (vIL-6). We show here that XBP-1s can also directly activate KSHV ORF21, a lytic gene. ORF21 encodes KSHV thymidine kinase (TK), which increases the pool of dTTP for viral replication and enhances lytic replication. Direct activation of ORF21 by XBP-1s can enhance viral replication in germinal center B cells and contribute to the pathogenesis of KSHV multicentric Castleman disease (MCD). KSHV-MCD is characterized by systemic inflammation caused, in part, by lytic replication and overproduction of KSHV vIL-6 in XBP-1s-expressing lymph node plasmablasts. KSHV thymidine kinase can phosphorylate zidovudine and ganciclovir to toxic moieties, and direct activation of ORF21 by XBP-1s may also help explain the effectiveness of zidovudine and valganciclovir in the treatment of KSHV-MCD.

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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

Journal of Virology ◽

10.1128/jvi.01003-16 ◽

2016 ◽

Vol 90 (19) ◽

pp. 8822-8841 ◽

Cited By ~ 36

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.

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Kv1.3 induced hyperpolarisation and Cav3.2-mediated calcium entry are required for efficient Kaposi’s sarcoma-associated herpesvirus lytic replication

10.1101/2021.09.10.459757 ◽

2021 ◽

Author(s):

Holli Carden ◽

Mark L. Dallas ◽

David J. Hughes ◽

Jonathan D. Lippiat ◽

Jamel Mankouri ◽

...

Keyword(s):

B Cell ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Host Factors ◽

Transcription Activator ◽

Intracellular Ca ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

First Time

AbstractUnderstanding the host factors critical for Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic replication can identify new targets for therapeutic intervention. Using pharmacological and genetic silencing approaches, we reveal for the first time that KSHV requires a B cell expressed voltage-gated K+ channel, Kv1.3, to enhance lytic replication. We show that the KSHV replication and transcription activator (RTA) protein upregulates Kv1.3 expression, leading to enhanced K+ channel activity and hyperpolarisation of the B cell membrane. Enhanced Kv1.3 activity then promotes intracellular Ca2+ influx through Cav3.2, a T-type Ca2+ channel, leading to the Ca2+ driven nuclear localisation of NFAT and the subsequent NFAT1-responsive gene expression. Importantly, KSHV lytic replication and infectious virion production could be inhibited by both Kv1.3 and Cav3.2 blockers or through Kv1.3 silencing. These findings provide new mechanistic insight into the essential role of host ion channels during KSHV infection and highlight Kv1.3 and Cav3.2 as new druggable host factors that are key to the successful completion of KSHV lytic replication.

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

Journal of Virology ◽

10.1128/jvi.00505-17 ◽

2017 ◽

Vol 91 (19) ◽

Cited By ~ 5

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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Enhancement of Autophagy during Lytic Replication by the Kaposi's Sarcoma-Associated Herpesvirus Replication and Transcription Activator

Journal of Virology ◽

10.1128/jvi.00024-10 ◽

2010 ◽

Vol 84 (15) ◽

pp. 7448-7458 ◽

Cited By ~ 46

Author(s):

Hui-Ju Wen ◽

Zhilong Yang ◽

You Zhou ◽

Charles Wood

Keyword(s):

Kaposi's Sarcoma ◽

Degradation Mechanism ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Transcription Activator ◽

Lytic Gene ◽

Lytic Reactivation ◽

Autophagic Process ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Autophagy is one of two major degradation systems in eukaryotic cells. The degradation mechanism of autophagy is required to maintain the balance between the biosynthetic and catabolic processes and also contributes to defense against invading pathogens. Recent studies suggest that a number of viruses can evade or subvert the host cell autophagic pathway to enhance their own replication. Here, we investigated the effect of autophagy on the KSHV (Kaposi's sarcoma-associated herpesvirus) life cycle. We found that the inhibition of autophagy reduces KSHV lytic reactivation from latency, and an enhancement of autophagy can be detected during KSHV lytic replication. In addition, RTA (replication and transcription activator), an essential viral protein for KSHV lytic reactivation, is able to enhance the autophagic process, leading to an increase in the number of autophagic vacuoles, an increase in the level of the lipidated LC3 protein, and the formation of autolysosomes. Moreover, the inhibition of autophagy affects RTA-mediated lytic gene expression and viral DNA replication. These results suggest that RTA increases autophagy activation to facilitate KSHV lytic replication. This is the first report demonstrating that autophagy is involved in the lytic reactivation of KSHV.

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X-Box Binding Protein 1 Contributes to Induction of the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle under Hypoxic Conditions

Journal of Virology ◽

10.1128/jvi.00076-09 ◽

2009 ◽

Vol 83 (14) ◽

pp. 7202-7209 ◽

Cited By ~ 39

Author(s):

Lucy Dalton-Griffin ◽

Sam J. Wilson ◽

Paul Kellam

Keyword(s):

Kaposi's Sarcoma ◽

Primary Effusion Lymphoma ◽

Binding Protein ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Lytic Cycle ◽

Response Element ◽

Hypoxic Conditions ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV), like other herpesviruses, has two stages to its life cycle: latency and lytic replication. KSHV is required for development of Kaposi's sarcoma, a tumor of endothelial origin, and is associated with the B-cell tumor primary effusion lymphoma (PEL) and the plasmablastic variant of multicentric Castleman's disease, all of which are characterized by predominantly latent KSHV infection. Recently, we and others have shown that the activated form of transcription factor X-box binding protein 1 (XBP-1) is a physiological trigger of KSHV lytic reactivation in PEL. Here, we show that XBP-1s transactivates the ORF50/RTA promoter though an ACGT core containing the XBP-1 response element, an element previously identified as a weakly active hypoxia response element (HRE). Hypoxia induces the KSHV lytic cycle, and active HREs that respond to hypoxia-inducible factor 1α are present in the ORF50/RTA promoter. Hypoxia also induces active XBP-1s, and here, we show that both transcription factors contribute to the induction of RTA expression, leading to the production of infectious KSHV under hypoxic conditions.

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