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

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.

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

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.

scholarly journals A Survey of the Interactome of Kaposi's Sarcoma-Associated Herpesvirus ORF45 Revealed Its Binding to Viral ORF33 and Cellular USP7, Resulting in Stabilization of ORF33 That Is Required for Production of Progeny Viruses

2015 ◽  
Vol 89 (9) ◽  
pp. 4918-4931 ◽  
Author(s):  
Joseph Gillen ◽  
Wenwei Li ◽  
Qiming Liang ◽  
Denis Avey ◽  
Jianjun Wu ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Binding Domain ◽  
Specific Protease ◽  
Infected Cells ◽  
Ubiquitin Specific Protease ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTThe ORF45 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus-specific immediate-early tegument protein. Our previous studies have revealed its crucial roles in both early and late stages of KSHV infection. In this study, we surveyed the interactome of ORF45 using a panel of monoclonal antibodies. In addition to the previously identified extracellular regulated kinase (ERK) and p90 ribosomal S6 kinase (RSK) proteins, we found several other copurified proteins, including prominent ones of ∼38 kDa and ∼130 kDa. Mass spectrometry revealed that the 38-kDa protein is viral ORF33 and the 130-kDa protein is cellular USP7 (ubiquitin-specific protease 7). We mapped the ORF33-binding domain to the highly conserved carboxyl-terminal 19 amino acids (aa) of ORF45 and the USP7-binding domain to the reported consensus motif in the central region of ORF45. Using immunofluorescence staining, we observed colocalization of ORF45 with ORF33 or USP7 both under transfected conditions and in KSHV-infected cells. Moreover, we noticed ORF45-dependent relocalization of a portion of ORF33/USP7 from the nucleus to the cytoplasm. We found that ORF45 caused an increase in ORF33 protein accumulation that was abolished if either the ORF33- or USP7-binding domain in ORF45 was deleted. Furthermore, deletion of the conserved carboxyl terminus of ORF45 in the KSHV genome drastically reduced the level of ORF33 protein in KSHV-infected cells and abolished production of progeny virions. Collectively, our results not only reveal new components of the ORF45 interactome, but also demonstrate that the interactions among these proteins are crucial for KSHV lytic replication.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several human cancers. KSHV ORF45 is a multifunctional protein that is required for KSHV lytic replication, but the exact mechanisms by which ORF45 performs its critical functions are unclear. Our previous studies revealed that all ORF45 protein in cells exists in high-molecular-weight complexes. We therefore sought to characterize the interactome of ORF45 to provide insights into its roles during lytic replication. Using a panel of monoclonal antibodies, we surveyed the ORF45 interactome in KSHV-infected cells. We identified two new binding partners of ORF45: the viral protein ORF33 and cellular ubiquitin-specific protease 7 (USP7). We further demonstrate that the interaction between ORF45 and ORF33 is crucial for the efficient production of KSHV viral particles, suggesting that the targeted interference with this interaction may represent a novel strategy to inhibit KSHV lytic replication.

scholarly journals The K1 Protein of Kaposi's Sarcoma-Associated Herpesvirus Augments Viral Lytic Replication

2016 ◽  
Vol 90 (17) ◽  
pp. 7657-7666 ◽  
Author(s):  
Zhigang Zhang ◽  
Wuguo Chen ◽  
Marcia K. Sanders ◽  
Kevin F. Brulois ◽  
Dirk P. Dittmer ◽  
...  
Keyword(s):  
Life Cycle ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Akt Kinase ◽  
Recombinant Viruses ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTThe K1 gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) is encoded by the first open reading frame (ORF) of the viral genome. To investigate the role of the K1 gene during the KSHV life cycle, we constructed a set of recombinant viruses that contained either wild-type (WT) K1, a deleted K1 ORF (KSHVΔK1), stop codons within the K1 ORF (KSHV-K15×STOP), or a revertant K1 virus (KSHV-K1REV). We report that the recombinant viruses KSHVΔK1 and KSHV-K15×STOPdisplayed significantly reduced lytic replication compared to WT KSHV and KSHV-K1REVupon reactivation from latency. Additionally, cells infected with the recombinant viruses KSHVΔK1 and KSHV-K15×STOPalso yielded smaller amounts of infectious progeny upon reactivation than did WT KSHV- and KSHV-K1REV-infected cells. Upon reactivation from latency, WT KSHV- and KSHV-K1REV-infected cells displayed activated Akt kinase, as evidenced by its phosphorylation, while cells infected with viruses deleted for K1 showed reduced phosphorylation and activation of Akt kinase. Overall, our results suggest that K1 plays an important role during the KSHV life cycle.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of three human malignancies, and KSHV K1 is a signaling protein that has been shown to be involved in cellular transformation and to activate the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway. In order to investigate the role of the K1 protein in the life cycle of KSHV, we constructed recombinant viruses that were deficient for K1. We found that K1 deletion viruses displayed reduced lytic replication compared to the WT virus and also yielded smaller numbers of infectious progeny. We report that K1 plays an important role in the life cycle of KSHV.

scholarly journals Asynchronous Progression through the Lytic Cascade and Variations in Intracellular Viral Loads Revealed by High-Throughput Single-Cell Analysis of Kaposi's Sarcoma-Associated Herpesvirus Infection

2006 ◽  
Vol 80 (20) ◽  
pp. 10073-10082 ◽  
Author(s):  
Laura A. Adang ◽  
Christopher H. Parsons ◽  
Dean H. Kedes
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
High Throughput ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Immediate Early ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus-8) is frequently tumorigenic in immunocompromised patients. The average intracellular viral copy number within infected cells, however, varies markedly by tumor type. Since the KSHV-encoded latency-associated nuclear antigen (LANA) tethers viral episomes to host heterochromatin and displays a punctate pattern by fluorescence microscopy, we investigated whether accurate quantification of individual LANA dots is predictive of intracellular viral genome load. Using a novel technology that integrates single-cell imaging with flow cytometry, we found that both the number and the summed immunofluorescence of individual LANA dots are directly proportional to the amount of intracellular viral DNA. Moreover, combining viral (immediate early lytic replication and transcription activator [RTA] and late lytic K8.1) and cellular (syndecan-1) staining with image-based flow cytometry, we were also able to rapidly and simultaneously distinguish among cells supporting latent, immediate early lytic, early lytic, late lytic, and a potential fourth “delayed late” category of lytic replication. Applying image-based flow cytometry to KSHV culture models, we found that de novo infection results in highly varied levels of intracellular viral load and that lytic induction of latently infected cells likewise leads to a heterogeneous population at various stages of reactivation. These findings additionally underscore the potential advantages of studying KSHV biology with high-throughput analysis of individual cells.

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

2011 ◽  
Vol 56 (2) ◽  
pp. 893-902 ◽  
Author(s):  
Lorenzo González-Molleda ◽  
Yan Wang ◽  
Yan Yuan
Keyword(s):  
Dna Replication ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Cellular Proteins ◽  
Virion Production ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Topo Ii ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Lytic Dna Replication

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.

scholarly journals The Viral Bcl-2 Homologs of Kaposi's Sarcoma-Associated Herpesvirus and Rhesus Rhadinovirus Share an Essential Role for Viral Replication

2017 ◽  
Vol 91 (6) ◽  
Author(s):  
Antonio Gallo ◽  
Melanie Lampe ◽  
Thomas Günther ◽  
Wolfram Brune
Keyword(s):  
Viral Replication ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Replication Cycle ◽  
Inhibiting Activity ◽  
Infected Cells ◽  
Essential Function ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT KS-Bcl-2 is a Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded viral Bcl-2 (vBcl-2) homolog which has apoptosis- and autophagy-inhibiting activity when expressed in transfected cells. However, little is known about its function during viral infection. As KS-Bcl-2 is expressed during the lytic replication cycle, we used constitutively lytic and inducibly lytic KSHV mutants to investigate the role of KS-Bcl-2 during the lytic cycle. We show that KSHV cannot complete the lytic replication cycle and produce infectious progeny in the absence of KS-Bcl-2, indicating that the protein is essential for KSHV replication. Replacement of the KS-Bcl-2 coding sequence, ORF16, by sequences encoding a potent cellular apoptosis and autophagy inhibitor, Bcl-XL, or the cytomegalovirus mitochondrial inhibitor of apoptosis, vMIA, did not rescue KSHV replication, suggesting that KS-Bcl-2 has a function that goes beyond apoptosis and autophagy inhibition. Strikingly, the vBcl-2 proteins of the related γ2-herpesviruses murine herpesvirus 68 and herpesvirus saimiri did not rescue the replication of a KS-Bcl-2 deletion mutant, but rhesus rhadinovirus (RRV) vBcl-2 did. Deletion of ORF16 from the RRV genome abrogated viral replication, but its replacement by KSHV ORF16 rescued RRV replication, indicating that the essential vBcl-2 function is conserved between these two primate rhadinoviruses. We further show that the KSHV and RRV Bcl-2 homologs localize to the mitochondria and nuclei of infected cells. Deletion of 17 amino acids from the N terminus of KS-Bcl-2 abrogates nuclear localization and KSHV replication, suggesting that KS-Bcl-2 might execute its essential function in the nuclei of infected cells. IMPORTANCE Several viruses express proteins homologous to cellular Bcl-2. Viral Bcl-2 proteins have functions similar to those of cellular Bcl-2: they can inhibit apoptosis, a form of programmed cell death, and autophagy, a self-degradative process for the disposal of dysfunctional or unwanted components. This study shows that the vBcl-2 proteins of KSHV and RRV differ from other vBcl-2 proteins in that they are essential for viral replication. The essential function is separate from the apoptosis- and autophagy-inhibiting activity but correlates with an unusual localization within the cell nucleus, suggesting that these proteins exert a novel function in the nucleus.

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

2006 ◽  
Vol 80 (11) ◽  
pp. 5251-5260 ◽  
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.

scholarly journals Transcriptional Origin of Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs

2006 ◽  
Vol 80 (5) ◽  
pp. 2234-2242 ◽  
Author(s):  
Xuezhong Cai ◽  
Bryan R. Cullen
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Open Reading Frames ◽  
Polyadenylation Signal ◽  
Latently Infected ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
The One ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 11 distinct microRNAs, all of which are found clustered within the major latency-associated region of the KSHV genome in the same transcriptional orientation. Because the KSHV microRNAs are all expressed in latently infected cells and are largely unaffected by induction of lytic replication, it appeared probable that they would be processed out of KSHV transcripts that are derived from a latent promoter(s) present in this region. Here, we define three latent transcripts, derived from two distinct KSHV latent promoters, that function as both KSHV primary microRNA precursors and as kaposin pre-mRNAs. These activities require the readthrough of a leaky viral polyadenylation signal located at nucleotide 122070 in the KSHV genome. In contrast, recognition of this polyadenylation signal gives rise to previously identified mRNAs that encode the KSHV open reading frames (ORFs) 71, 72 and 73 proteins as well as a novel unspliced KSHV mRNA that encodes only ORF72 and ORF71. Thus, transcripts initiating at the two latent promoters present in the KSHV latency-associated region can undergo two entirely distinct fates, i.e., processing to give a kaposin mRNA and viral microRNAs on the one hand or expression as KSHV ORF71, ORF72, or ORF73 mRNAs on the other, depending on whether the viral polyadenylation site located at position 122070 is ignored or recognized, respectively.

scholarly journals 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

2009 ◽  
Vol 83 (10) ◽  
pp. 5056-5066 ◽  
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.

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

2006 ◽  
Vol 80 (24) ◽  
pp. 12171-12186 ◽  
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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