Potent Antiviral Activity of Topoisomerase I and II Inhibitors against Kaposi's Sarcoma-Associated Herpesvirus

ABSTRACTThe lytic DNA replication of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates at an origin (ori-Lyt) and requirestrans-acting elements, both viral and cellular. We recently demonstrated that several host cellular proteins, including topoisomerases I and II (Topo I and II), are involved in KSHV lytic DNA replication (Y. Wang, H. Li, Q. Tang, G. G. Maul, and Y. Yuan. J. Virol. 82: 2867–2882, 2008). To assess the importance of these topoisomerases in viral lytic replication, shRNA-mediated gene silencing was used. Depletion of Topo I and II severely inhibited viral lytic DNA replication as well as virion production, suggesting essential roles of these cellular proteins in viral DNA replication. The discovery of Topo I and II as enzymes indispensable for KSHV DNA replication raises a possibility that these cellular proteins could be new targets of therapeutic approaches to halt KSHV replication and treat KSHV-associated diseases. In this report, we examined one Topo I inhibitor and several Topo II inhibitors (inclusive of Topo II poison and catalytic inhibitors) as potential therapeutic agents for blocking KSHV replication. The Topo II catalytic inhibitors in general exhibited marked inhibition on KSHV replication and minimal cytotoxicity. In particular, novobiocin, with the best selectivity index (SI = 31.62) among the inhibitors tested in this study, is effective in inhibiting KSHV DNA replication and virion production but shows little adverse effect on cell proliferation and cycle progression in its therapeutic concentration, suggesting its potential to become an effective and safe drug for the treatment of human diseases associated with KSHV 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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Kaposi's Sarcoma-Associated Herpesvirus ori-Lyt-Dependent DNA Replication: Involvement of Host Cellular Factors

Journal of Virology ◽

10.1128/jvi.01319-07 ◽

2008 ◽

Vol 82 (6) ◽

pp. 2867-2882 ◽

Cited By ~ 57

Author(s):

Yan Wang ◽

Hong Li ◽

Qiyi Tang ◽

Gerd G. Maul ◽

Yan Yuan

Keyword(s):

Dna Replication ◽

Viral Replication ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Cellular Proteins ◽

Cellular Factors ◽

Virally Encoded ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Lytic Dna Replication ◽

Parp 1

ABSTRACT Herpesvirus lytic DNA replication requires both the cis-acting element, the origin, and trans-acting factors, including virally encoded origin-binding protein, DNA replication enzymes, and auxiliary factors. Two lytic DNA replication origins (ori-Lyt) of Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified, and two virally encoded proteins, namely, RTA and K8, have been shown to bind to the origins. In this study, we sought to identify cellular factors that associate with ori-Lyt by using DNA affinity purification and mass spectrometry. This approach led to identification of several cellular proteins that bind to KSHV ori-Lyt. They include topoisomerases (Topo) I and II, MSH2/6, RecQL, poly(ADP-ribose) polymerase I (PARP-1), DNA-PK, Ku86/70 autoantigens, and scaffold attachment factor A (SAF-A). RecQL appears to associate with prereplication complexes and be recruited to ori-Lyt through RTA and K8. Topoisomerases, MSH2, PARP-1, DNA-PK, and Ku86 were not detected in prereplication complexes but were present in replication initiation complexes on ori-Lyt. All these cellular proteins accumulate in viral replication compartments in the nucleus, indicating that these proteins may have a role in viral replication. Topo I and II appear to be essential for viral DNA replication as inhibition of their activities with specific inhibitors (camptothecin and ellipticine) blocked ori-Lyt-dependent DNA replication. Furthermore, inhibition of PARP-1 with chemical inhibitors (3-aminobenzamide and niacinamide) resulted in decreased ori-Lyt-dependent DNA replication, whereas hydroxyurea, which raises PARP-1 activity, caused an increase in the DNA replication, suggesting a positive role for PARP-1 in KSHV lytic DNA replication.

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HMGB1 knockout decreases Kaposi's sarcoma-associated herpesvirus virion production in iSLK BAC16 cells by attenuating viral gene expression

Journal of Virology ◽

10.1128/jvi.00799-21 ◽

2021 ◽

Author(s):

Su-Kyung Kang ◽

Yun Hee Kang ◽

Seung-Min Yoo ◽

Changhoon Park ◽

Hong Seok Kim ◽

...

Keyword(s):

Gene Expression ◽

Kaposi's Sarcoma ◽

High Mobility ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Virion Production ◽

Viral Genes ◽

Infected Cells ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

Multiple host proteins affect the gene expression of Kaposi's sarcoma-associated herpesvirus (KSHV) during latent and lytic replication. The high mobility group box 1 (HMGB1) serves as a highly conserved chromosomal protein inside the cell and a prototypical damage-associated molecular pattern molecule outside the cell. HMGB1 has been shown to play a pathogenic role in viral infectious diseases and to regulate the lytic replication of KSHV. However, its functional effects on the KSHV life cycle in KSHV-infected cells have not been fully elucidated. Here, we explored the role of the intracellular and extracellular HMGB1 in KSHV virion production by employing CRISPR/Cas9-mediated HMGB1 knockout in the KSHV-producing iSLK BAC16 cell line. Intracellular HMGB1 formed complexes with various proteins, and the abundance of HMGB1-interacting proteins changed during latent and lytic replication. Moreover, extracellular HMGB1 was found to enhance lytic replication by phosphorylating JNK. Of note, the expression of viral genes was attenuated during lytic replication in HMGB1- knockout iSLK BAC16 cells, with significantly decreased production of infectious virions compared to that in wild-type cells. Collectively, our results demonstrate that HMGB1 is an important cellular cofactor that affects the generation of infectious KSHV progeny during lytic replication. Author Summary The high mobility group box 1 protein ( HMGB1 ) has many intra- and extracellular biological functions with an intricate role in various diseases. In certain viral infections, HMGB1 affects the viral life cycle and pathogenesis. In this study, we explored the effects of HMGB1 knockout on the production of Kaposi’s sarcoma-associated herpesvirus (KSHV). HMGB1 knockout decreased virion production in KSHV-producing cells by decreasing the expression of viral genes. The processes by which HMGB1 affects KSHV production may occur inside or outside of infected cells. For instance, several cellular and viral proteins interacted with intracellular HMGB1 in a nucleosomal complex; whereas extracellular HMGB1 induced JNK phosphorylation, thus enhancing lytic replication. Our results suggest that both intracellular and extracellular HMGB1 are necessary for efficient KSHV replication. Thus, HMGB1 may represent an effective therapeutic target for the regulation of KSHV production.

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Kaposi's Sarcoma-Associated Herpesvirus Lytic Gene ORF57 Is Essential for Infectious Virion Production

Journal of Virology ◽

10.1128/jvi.02570-05 ◽

2006 ◽

Vol 80 (11) ◽

pp. 5251-5260 ◽

Cited By ~ 61

Author(s):

Zhao Han ◽

Sankar Swaminathan

Keyword(s):

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Lytic Gene ◽

Barr Virus ◽

Virion Production ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Epstein Barr ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The ORF57 gene of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a nuclear protein expressed during the lytic phase of KSHV replication. An ORF57 homolog is present in all known human herpesviruses and many animal herpesviruses. Many of these proteins have been demonstrated to have essential transcriptional and posttranscriptional regulatory functions. ORF57 enhances expression of reporter genes posttranscriptionally in vitro and may synergize with transcription factors to enhance gene transcription. However, the biologic role of ORF57 in KSHV replication has not been established. In this study, we demonstrate that ORF57 is essential for productive KSHV lytic replication by constructing a recombinant KSHV in which ORF57 expression has been specifically inactivated. The ORF57-null KSHV recombinant was unable to produce virion progeny or fully express several other lytic KSHV genes except when ORF57 was provided in trans. The Epstein-Barr virus (EBV) homolog of ORF57, SM, was unable to rescue lytic KSHV virion production, although EBV SM does enhance KSHV lytic gene expression from the ORF57-null mutant. Conversely, ORF57 did not rescue an SM-null recombinant EBV, indicating the existence of virus-specific functions for the ORF57 family of genes.

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Requirement of a 12-Base-Pair TATT-Containing Sequence and Viral Lytic DNA Replication in Activation of the Kaposi's Sarcoma-Associated Herpesvirus K8.1 Late Promoter

Journal of Virology ◽

10.1128/jvi.78.5.2609-2614.2004 ◽

2004 ◽

Vol 78 (5) ◽

pp. 2609-2614 ◽

Cited By ~ 37

Author(s):

Shuang Tang ◽

Koji Yamanegi ◽

Zhi-Ming Zheng

Keyword(s):

Dna Replication ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Late Promoter ◽

Viral Dna Replication ◽

Origin Of Dna Replication ◽

Infected Cells ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Lytic Dna Replication

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) K8.1 late promoter consists of a minimal 24-bp sequence, with a TATA-like, 12-bp promoter core, AATATTAAAGGG, and is active on a reporter only in butyrate-induced KSHV-infected cells. The activity of the K8.1 promoter can be enhanced (>15-fold) by the KSHV left-end lytic origin of DNA replication (oriLyt-L) sequence while providing inefficient replication of plasmid DNA and is inhibited by viral DNA replication inhibitors, suggesting that activation of the K8.1 promoter on the reporter is involved in KSHV lytic DNA replication largely by trans.

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Dimethyl Sulfoxide Enhances Kaposi’s Sarcoma-Associated Herpesvirus Production During Lytic Replication

Frontiers in Microbiology ◽

10.3389/fmicb.2021.778525 ◽

2021 ◽

Vol 12 ◽

Author(s):

Su-Kyung Kang ◽

Myung-Ju Lee ◽

Ho-Hyun Ryu ◽

Jisu Lee ◽

Myung-Shin Lee

Keyword(s):

Dimethyl Sulfoxide ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Viral Production ◽

Virion Production ◽

Dmso Treatment ◽

Infected Cells ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an etiologic agent of Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman disease. In studies of KSHV, efficient virus production and isolation are essential. Reactivation of KSHV can be initiated by treating latently infected cells with chemicals, such as 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate. These chemicals have been used as tools to induce lytic replication and viral production in KSHV-producing cell lines. Dimethyl sulfoxide (DMSO) is an organosulfur compound that is frequently used as an aprotic solvent similar to water. In experiments exploring signaling pathways in KSHV-infected cells, DMSO treatment alone as a vehicle affected the lytic gene expression of KSHV. However, to the best of our knowledge, the effects of DMSO on KSHV-producing cells have not yet been reported. Therefore, in this study, we investigated whether DMSO could be used as a reagent to enhance viral production during lytic replication in KSHV-producing cells and assessed the underlying mechanisms. The effects of DMSO on KSHV production were analyzed in iSLK BAC16 cells, which have been widely used for recombinant KSHV production. We found that the production of KSHV virions was significantly increased by treatment with DMSO during the induction of lytic replication. Mechanistically, lytic genes of KSHV were enhanced by DMSO treatment, which was correlated with virion production. Additionally, DMSO induced the phosphorylation of JNK during lytic replication, and inhibition of JNK abolished the effects of DMSO on lytic replication and virion production. Our findings showed that additional treatment with DMSO during the induction of lytic replication significantly improved the yield of KSHV production.

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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 Latency-Associated Nuclear Antigen Induces a Strong Bend on Binding to Terminal Repeat DNA

Journal of Virology ◽

10.1128/jvi.79.21.13829-13836.2005 ◽

2005 ◽

Vol 79 (21) ◽

pp. 13829-13836 ◽

Cited By ~ 22

Author(s):

Lai-Yee Wong ◽

Angus C. Wilson

Keyword(s):

Dna Replication ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Nuclear Antigen ◽

Major Groove ◽

Repeat Dna ◽

Initiation Of Dna Replication ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Herpesvirus Genome

ABSTRACT During latency, the Kaposi's sarcoma-associated herpesvirus genome is maintained as a circular episome, replicating in synchrony with host chromosomes. Replication requires the latency-associated nuclear antigen (LANA) and an origin of latent DNA replication located in the viral terminal repeats, consisting of two LANA binding sites (LBSs) and a GC-rich sequence. Here, we show that the recruitment of a LANA dimer to high-affinity site LBS-1 bends DNA by 57° and towards the major groove. The cooccupancy of LBS-1 and lower-affinity LBS-2 induces a symmetrical bend of 110°. By changing the origin architecture, LANA may help to assemble a specific nucleoprotein structure important for the initiation of DNA replication.

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Bub1 in Complex with LANA Recruits PCNA To Regulate Kaposi's Sarcoma-Associated Herpesvirus Latent Replication and DNA Translesion Synthesis

Journal of Virology ◽

10.1128/jvi.01524-15 ◽

2015 ◽

Vol 89 (20) ◽

pp. 10206-10218 ◽

Cited By ~ 13

Author(s):

Zhiguo Sun ◽

Hem Chandra Jha ◽

Erle S. Robertson

Keyword(s):

Dna Replication ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Kinase Domain ◽

Cellular Protein ◽

Nuclear Antigen ◽

Infected Cells ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Molecular Bridge

ABSTRACTLatent DNA replication of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates at the terminal repeat (TR) element and requirestrans-acting elements, both viral and cellular, such as ORCs, MCMs, and latency-associated nuclear antigen (LANA). However, how cellular proteins are recruited to the viral genome is not very clear. Here, we demonstrated that the host cellular protein, Bub1, is involved in KSHV latent DNA replication. We show that Bub1 constitutively interacts with proliferating cell nuclear antigen (PCNA) via a highly conserved PIP box motif within the kinase domain. Furthermore, we demonstrated that Bub1 can form a complex with LANA and PCNA in KSHV-positive cells. This strongly indicated that Bub1 serves as a scaffold or molecular bridge between LANA and PCNA. LANA recruited PCNA to the KSHV genome via Bub1 to initiate viral replication in S phase and interacted with PCNA to promote its monoubiquitination in response to UV-induced damage for translesion DNA synthesis. This resulted in increased survival of KSHV-infected cells.IMPORTANCEDuring latency in KSHV-infected cells, the viral episomal DNA replicates once each cell cycle. KSHV does not express DNA replication proteins during latency. Instead, KSHV LANA recruits the host cell DNA replication machinery to the replication origin. However, the mechanism by which LANA mediates replication is uncertain. Here, we show that LANA is able to form a complex with PCNA, a critical protein for viral DNA replication. Furthermore, our findings suggest that Bub1, a spindle checkpoint protein, serves as a scaffold or molecular bridge between LANA and PCNA. Our data further support a role for Bub1 and LANA in PCNA-mediated cellular DNA replication processes as well as monoubiquitination of PCNA in response to UV damage. These data reveal a therapeutic target for inhibition of KSHV persistence in malignant cells.

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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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