Kaposi's Sarcoma Associated Herpesvirus Tegument Protein ORF75 Is Essential for Viral Lytic Replication and Plays a Critical Role in the Antagonization of ND10-Instituted Intrinsic Immunity

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). We previously reported that hypoxia activates KSHV lytic replication and that the promoter for open reading frame 34 (ORF34) contains a functional hypoxia-responsive element (HRE). ORF34 is part of a cluster of lytic genes (ORF34-37) that includes ORF36, a phosphotransferase, and ORF37, a shutoff exonuclease. Rapid amplification of cDNA ends analysis revealed that they share a common polyadenylation signal but have two start sites. Two transcripts were identified, one 3.4 kb encoding ORF35-37, and the other 4.2 kb encoding ORF34 and also having coding potential for ORF35-37. Exposure of PEL cell lines to hypoxia induced messages of lengths consistent with those of these transcripts. Reporter assays with Hep3B cells showed activation of both transcripts by hypoxia. The ORF34-37 promoter region has six consensus HREs. Sequential deletion, site-directed mutagenesis experiments, and Northern blot analysis of RNA produced by constructs indicated that the second HRE (HRE-2) plays a critical role in the hypoxic activation of both RNA transcripts. The ORF35-37 transcript was upregulated by cotransfected hypoxia-inducible factor (HIF). Electrophoretic mobility shift assays demonstrated that HRE-2 and ancillary sequences bind and compete for HIF with hypoxic Hep3B nuclear extract. The activation of this gene cluster by hypoxia may have implications for the pathogenesis of PEL and KS. Moreover, the activation of ORF36 by hypoxia might be exploited to develop targeted therapy for PEL, which arises in a hypoxic environment (pleural effusions).

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FoxO1 Suppresses Kaposi’s Sarcoma-Associated Herpesvirus Lytic Replication and Controls Viral Latency

Journal of Virology ◽

10.1128/jvi.01681-18 ◽

2018 ◽

Vol 93 (3) ◽

Cited By ~ 4

Author(s):

Ruoyun Gao ◽

Tingting Li ◽

Brandon Tan ◽

Suzane Ramos da Silva ◽

Jae U. Jung ◽

...

Keyword(s):

Kaposi's Sarcoma ◽

Critical Role ◽

Kaposi’S Sarcoma ◽

Oxygen Free Radical ◽

Free Radical Scavenger ◽

Lytic Replication ◽

Radical Scavenger ◽

Intracellular Ros ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) has latent and lytic replication phases, both of which contribute to the development of KSHV-induced malignancies. Among the numerous factors identified to regulate the KSHV life cycle, oxidative stress, caused by imbalanced clearing and production of reactive oxygen species (ROS), has been shown to robustly disrupt KSHV latency and induce viral lytic replication. In this study, we identified an important role of the antioxidant defense factor forkhead box protein O1 (FoxO1) in the KSHV life cycle. Either chemical inhibition of the FoxO1 function or knockdown of FoxO1 expression led to an increase in the intracellular ROS level that was subsequently sufficient to disrupt KSHV latency and induce viral lytic reactivation. On the other hand, treatment with N-acetyl-l-cysteine (NAC), an oxygen free radical scavenger, led to a reduction in the FoxO1 inhibition-induced ROS level and, ultimately, the attenuation of KSHV lytic reactivation. These findings reveal that FoxO1 plays a critical role in keeping KSHV latency in check by maintaining the intracellular redox balance. IMPORTANCE Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with several cancers, including Kaposi’s sarcoma (KS). Both the KSHV latent and lytic replication phases are important for the development of KS. Identification of factors regulating the KSHV latent phase-to-lytic phase switch can provide insights into the pathogenesis of KSHV-induced malignancies. In this study, we show that the antioxidant defense factor forkhead box protein O1 (FoxO1) maintains KSHV latency by suppressing viral lytic replication. Inhibition of FoxO1 disrupts KSHV latency and induces viral lytic replication by increasing the intracellular ROS level. Significantly, treatment with an oxygen free radical scavenger, N-acetyl-l-cysteine (NAC), attenuated the FoxO1 inhibition-induced intracellular ROS level and KSHV lytic replication. Our works reveal a critical role of FoxO1 in suppressing KSHV lytic replication, which could be targeted for antiviral therapy.

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Kaposi's Sarcoma-Associated Herpesvirus Inhibitor of cGAS (KicGAS), Encoded by ORF52, Is an Abundant Tegument Protein and Is Required for Production of Infectious Progeny Viruses

Journal of Virology ◽

10.1128/jvi.02675-15 ◽

2016 ◽

Vol 90 (11) ◽

pp. 5329-5342 ◽

Cited By ~ 19

Author(s):

Wenwei Li ◽

Denis Avey ◽

Bishi Fu ◽

Jian-jun Wu ◽

Siming Ma ◽

...

Keyword(s):

Life Cycle ◽

Immune Evasion ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Tegument Protein ◽

Tegument Proteins ◽

Viral Particles ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTAlthough Kaposi's sarcoma-associated herpesvirus (KSHV) ORF52 (also known as KSHV inhibitor of cGAS [KicGAS]) has been detected in purified virions, the roles of this protein during KSHV replication have not been characterized. Using specific monoclonal antibodies, we revealed that ORF52 displays true late gene expression kinetics and confirmed its cytoplasmic localization in both transfected and KSHV-infected cells. We demonstrated that ORF52 comigrates with other known virion proteins following sucrose gradient centrifugation. We also determined that ORF52 resides inside the viral envelope and remains partially associated with capsid when extracellular virions are treated with various detergents and/or salts. There results indicate that ORF52 is a tegument protein abundantly present in extracellular virions. To characterize the roles of ORF52 in the KSHV life cycle, we engineered a recombinant KSHV ORF52-null mutant virus and found that loss of ORF52 results in reduced virion production and a further defect in infectivity. Upon analysis of the virion composition of ORF52-null viral particles, we observed a decrease in the incorporation of ORF45, as well as other tegument proteins, suggesting that ORF52 is important for the packaging of other virion proteins. In summary, our results indicate that, in addition to its immune evasion function, KSHV ORF52 is required for the optimal production of infectious virions, likely due to its roles in virion assembly as a tegument protein.IMPORTANCEThe tegument proteins of herpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), play key roles in the viral life cycle. Each of the three subfamilies of herpesviruses (alpha, beta, and gamma) encode unique tegument proteins with specialized functions. We recently found that one such gammaherpesvirus-specific protein, ORF52, has an important role in immune evasion during KSHV primary infection, through inhibition of the host cytosolic DNA sensing pathway. In this report, we further characterize ORF52 as a tegument protein with vital roles during KSHV lytic replication. We found that ORF52 is important for the production of infectious viral particles, likely through its role in virus assembly, a critical process for KSHV replication and pathogenesis. More comprehensive investigation of the functions of tegument proteins and their roles in viral replication may reveal novel targets for therapeutic interventions against KSHV-associated diseases.

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Kaposi's Sarcoma-Associated Herpesvirus-Encoded Latency-Associated Nuclear Antigen Inhibits Lytic Replication by Targeting Rta: a Potential Mechanism for Virus-Mediated Control of Latency

Journal of Virology ◽

10.1128/jvi.78.12.6585-6594.2004 ◽

2004 ◽

Vol 78 (12) ◽

pp. 6585-6594 ◽

Cited By ~ 146

Author(s):

Ke Lan ◽

Daniel A. Kuppers ◽

Subhash C. Verma ◽

Erle S. Robertson

Keyword(s):

Kaposi's Sarcoma ◽

Critical Role ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Nuclear Antigen ◽

Viral Progeny ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Like other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV, also designated human herpesvirus 8) can establish a latent infection in the infected host. During latency a small number of genes are expressed. One of those genes encodes latency-associated nuclear antigen (LANA), which is constitutively expressed in cells during latent as well as lytic infection. LANA has previously been shown to be important for the establishment of latent episome maintenance through tethering of the viral genome to the host chromosomes. Under specific conditions, KSHV can undergo lytic replication, with the production of viral progeny. The immediate-early Rta, encoded by open reading frame 50 of KSHV, has been shown to play a critical role in switching from viral latent replication to lytic replication. Overexpression of Rta from a heterologous promoter is sufficient for driving KSHV lytic replication and the production of viral progeny. In the present study, we show that LANA down-modulates Rta's promoter activity in transient reporter assays, thus repressing Rta-mediated transactivation. This results in a decrease in the production of KSHV progeny virions. We also found that LANA interacts physically with Rta both in vivo and in vitro. Taken together, our results demonstrate that LANA can inhibit viral lytic replication by inhibiting expression as well as antagonizing the function of Rta. This suggests that LANA may play a critical role in maintaining latency by controlling the switch between viral latency and lytic replication.

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A Kaposi's Sarcoma-Associated Herpesvirus Protein That Forms Inhibitory Complexes with Type I Interferon Receptor Subunits, Jak and STAT Proteins, and Blocks Interferon-Mediated Signal Transduction

Journal of Virology ◽

10.1128/jvi.02516-08 ◽

2009 ◽

Vol 83 (10) ◽

pp. 5056-5066 ◽

Cited By ~ 42

Author(s):

Sabine A. Bisson ◽

Anne-Laure Page ◽

Don Ganem

Keyword(s):

Kaposi's Sarcoma ◽

Human Tumor ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Type I Interferons ◽

Type I ◽

Type I Ifn ◽

Reading Frame ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Type I interferons (IFNs) are important mediators of innate antiviral defense and function by activating a signaling pathway through their cognate type I receptor (IFNAR). Here we report that lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) efficiently blocks type I IFN signaling and that an important effector of this blockade is the viral protein RIF, the product of open reading frame 10. RIF blocks IFN signaling by formation of inhibitory complexes that contain IFNAR subunits, the Janus kinases Jak1 and Tyk2, and the STAT2 transcription factor. Activation of both Tyk2 and Jak1 is inhibited, and abnormal recruitment of STAT2 to IFNAR1 occurs despite the decrement in Tyk2 activity. As a result of these actions, phosphorylation of both STAT2 and STAT1 is impaired, with subsequent failure of ISGF3 accumulation in the nucleus. The presence in the viral genome of potent inhibitors of type I IFN signaling, along with several viral genes that block IFN induction, highlights the importance of the IFN pathway in the control of this human tumor virus infection.

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Kaposi's Sarcoma-Associated Herpesvirus ori-Lyt-Dependent DNA Replication: DualRole of Replication and Transcription Activator

Journal of Virology ◽

10.1128/jvi.00990-06 ◽

2006 ◽

Vol 80 (24) ◽

pp. 12171-12186 ◽

Cited By ~ 51

Author(s):

Yan Wang ◽

Qiyi Tang ◽

Gerd G. Maul ◽

Yan Yuan

Keyword(s):

Dna Replication ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Transcription Activator ◽

Binding Motifs ◽

Viral Propagation ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Lytic Dna Replication

ABSTRACT Lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for viral propagation and pathogenicity. In Kaposi's sarcoma lesions, constant lytic replication plays a role in sustaining the population of latently infected cells that otherwise are quickly lost by segregation of latent viral episomes as spindle cells divide. Lytic DNA replication initiates from an origin (ori-Lyt) and requires trans-acting elements. Two functional ori-Lyts have been identified in the KSHV genome. Some cis-acting and trans-acting elements for ori-Lyt-dependent DNA replication have been found. Among these, K8 binding sites, a cluster of C/EBP binding motifs, and a replication and transcription activator (RTA) responsive element (RRE) are crucial cis-acting elements. Binding of K8 and RTA proteins to these motifs in ori-Lyt DNA was demonstrated to be absolutely essential for DNA replication. In the present study, functional roles of RTA in ori-Lyt-dependent DNA replication have been investigated. Two distinct functions of RTA were revealed. First, RTA activates an ori-Lyt promoter and initiates transcription across GC-rich tandem repeats. This RTA-mediated transcription is indispensable for DNA replication. Second, RTA is a component of the replication compartment, where RTA interacts with prereplication complexes composed of at least six core machinery proteins and K8. The prereplication complexes are recruited to ori-Lyt DNA through RTA, which interacts with the RRE, as well as K8, which binds to a cluster of C/EBP binding motifs with the aid of C/EBP α. The revelation of these two functions of RTA, together with its role in initiation of a transcriptional cascade that leads to transcription of all viral lytic genes, shows that RTA is a critical initiator and regulator of KSHV lytic DNA replication and viral propagation.

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Lytic Replication-Defective Kaposi's Sarcoma-Associated Herpesvirus: Potential Role in Infection and Malignant Transformation

Journal of Virology ◽

10.1128/jvi.78.20.11108-11120.2004 ◽

2004 ◽

Vol 78 (20) ◽

pp. 11108-11120 ◽

Cited By ~ 14

Author(s):

Jian-Hong Deng ◽

Yan-Jin Zhang ◽

Xin-Ping Wang ◽

Shou-Jiang Gao

Keyword(s):

Malignant Transformation ◽

Cell Lines ◽

Kaposi's Sarcoma ◽

Potential Role ◽

Primary Effusion Lymphoma ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Screening Methods ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Defective viruses often have pivotal roles in virus-induced diseases. Although Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), defective KSHV has not been reported. Using differential genetic screening methods, we show that defective KSHV is present in KS tumors and PEL cell lines. To investigate the role of defective viruses in KSHV-induced pathogenesis, we isolated and characterized a lytic replication-defective KSHV, KV-1, containing an 82-kb genomic deletion of solely lytic genes. Cells harboring KV-1 escaped G0/G1 apoptosis induced by spontaneous lytic replication occurred in cells infected with regular KSHV but maintained efficient latent replication. Consequently, KV-1-infected cells had phenotypes of enhanced cell proliferation and transformation potentials. Importantly, KV-1 was packaged as infectious virions by using regular KSHV as helpers, and KV-1-like variants were detected in cultures of two of five KSHV cell lines and 1 of 18 KS tumors. These results point to a potential role for defective viruses in the regulation of KSHV infection and malignant transformation.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01126-12 ◽

2012 ◽

Vol 56 (11) ◽

pp. 5794-5803 ◽

Cited By ~ 27

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.

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

Journal of Virology ◽

10.1128/jvi.02209-08 ◽

2008 ◽

Vol 83 (6) ◽

pp. 2531-2539 ◽

Cited By ~ 22

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.

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Activation of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Lytic Replication by Human Cytomegalovirus

Journal of Virology ◽

10.1128/jvi.75.3.1378-1386.2001 ◽

2001 ◽

Vol 75 (3) ◽

pp. 1378-1386 ◽

Cited By ~ 134

Author(s):

Jeffrey Vieira ◽

Patricia O'Hearn ◽

Louise Kimball ◽

Bala Chandran ◽

Lawrence Corey

Keyword(s):

Human Cytomegalovirus ◽

Kaposi's Sarcoma ◽

Human Fibroblasts ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Lytic Cycle ◽

Nuclear Antigen ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The majority of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells identified in vivo contain latent KSHV, with lytic replication in only a few percent of cells, as is the case for the cells of Kaposi's sarcoma (KS) lesions. Factors that influence KSHV latent or lytic replication are not well defined. Because persons with KS are often immunosuppressed and susceptible to many infectious agents, including human cytomegalovirus (HCMV), we have investigated the potential for HCMV to influence the replication of KSHV. Important to this work was the construction of a recombinant KSHV, rKSHV.152, expressing the green fluorescent protein (GFP) andneo (conferring resistance to G418). The expression of GFP was a marker of KSHV infection in cells of both epithelial and endothelial origin. The rKSHV.152 virus was used to establish cells, including human fibroblasts (HF), containing only latent KSHV, as demonstrated by latency-associated nuclear antigen expression and Gardella gel analysis. HCMV infection of KSHV latently infected HF activated KSHV lytic replication with the production of infectious KSHV. Dual-color immunofluorescence detected both the KSHV lytic open reading frame 59 protein and the HCMV glycoprotein B in coinfected cells, and UV-inactivated HCMV did not activate the production of infectious KSHV-GFP. In addition, HCMV coinfection increased the production of KSHV from endothelial cells and activated lytic cycle gene expression in keratinocytes. These data demonstrate that HCMV can activate KSHV lytic replication and suggest that HCMV could influence KSHV pathogenesis.

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