scholarly journals Kaposi's Sarcoma-Associated Herpesvirus ori-Lyt-Dependent DNA Replication: cis-Acting Requirements for Replication and ori-Lyt-Associated RNA Transcription

2004 ◽  
Vol 78 (16) ◽  
pp. 8615-8629 ◽  
Author(s):  
Yan Wang ◽  
Hong Li ◽  
Man Yee Chan ◽  
Fan Xiu Zhu ◽  
David M. Lukac ◽  
...  
Keyword(s):  
Dna Replication ◽  
Kaposi's Sarcoma ◽  
Binding Protein ◽  
Kaposi’S Sarcoma ◽  
Tata Box ◽  
Bzip Protein ◽  
Virally Encoded ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Lytic Dna Replication ◽  
Replication Function

ABSTRACT Herpesvirus lytic DNA replication requires both the cis-acting element, the origin, and trans-acting factors such as virally encoded origin-binding protein and DNA replication enzymes. Recently, the origins of lytic DNA replication (ori-Lyt) in Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified and a virally encoded bZip protein, K8, has been shown to specifically bind to the origin. To map cis-acting elements within KSHV ori-Lyt that are required for DNA replication function and to define the nature of K8 bZip protein binding to the origin, we constructed consecutive internal deletion mutations across the core domain of a KSHV ori-Lyt and tested them for DNA replication function in a transient replication assay. This mutagenesis study allowed the identification of four components within the ori-Lyt, and all were indispensable for ori-Lyt function. The first component contains eight CCAAT/enhancer binding protein (C/EBP) binding motifs that organize as four spaced C/EBP palindromes. Each palindrome contains two head-to-head CCAAT consensus motifs that are separated by a 13- or 12-bp space sequence. Substitution mutagenesis of these C/EBP motifs showed that these C/EBP palindromes are required for both K8 binding and ori-Lyt-dependent DNA replication. The second component is an 18-bp AT palindrome, which is essential for ori-Lyt function. The third component was determined to be a 32-bp previously unidentified sequence and is required for DNA replication. The last component consists of an open reading frame 50 (ORF50)/Rta responsive element (RRE) and a TATA box. We showed that the binding of an ORF50/Rta protein to the RRE was essential for ori-Lyt-dependent DNA replication. The presence of a functional RRE and a downstream TATA box suggested that this region serves as an ORF50/Rta-dependent promoter and a transcription event may be necessary for ori-Lyt-dependent DNA replication. Using a luciferase reporter system, we demonstrated that the region of the RRE and TATA box constitutes an ORF50/Rta-dependent promoter. Furthermore, a polyadenylated RNA of 1.4 kb was identified downstream of the promoter.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus ori-Lyt-Dependent DNA Replication: Involvement of Host Cellular Factors

2008 ◽  
Vol 82 (6) ◽  
pp. 2867-2882 ◽  
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.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Lytic Origin (ori-Lyt)-Dependent DNA Replication: Identification of the ori-Lyt and Association of K8 bZip Protein with the Origin

2003 ◽  
Vol 77 (10) ◽  
pp. 5578-5588 ◽  
Author(s):  
Cui Li Lin ◽  
Hong Li ◽  
Yan Wang ◽  
Fan Xiu Zhu ◽  
Sagar Kudchodkar ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Sequences ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Cycle ◽  
Replication Machinery ◽  
Bzip Protein ◽  
Virally Encoded ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Herpesviruses utilize different origins of replication during lytic versus latent infection. Latent DNA replication depends on host cellular DNA replication machinery, whereas lytic cycle DNA replication requires virally encoded replication proteins. In lytic DNA replication, the lytic origin (ori-Lyt) is bound by a virus-specified origin binding protein (OBP) that recruits the core replication machinery. In this report, we demonstrated that DNA sequences in two noncoding regions of the Kaposi's sarcoma-associated herpesvirus (KSHV) genome, between open reading frames (ORFs) K4.2 and K5 and between K12 and ORF71, are able to serve as origins for lytic cycle-specific DNA replication. The two ori-Lyt domains share an almost identical 1,153-bp sequence and a 600-bp downstream GC-rich repeat sequence, and the 1.7-kb DNA sequences are sufficient to act as a cis signal for replication. We also showed that an AT-palindromic sequence in the ori-Lyt domain is essential for the DNA replication. In addition, a virally encoded bZip protein, namely K8, was found to bind to a DNA sequence within the ori-Lyt by using a DNA binding site selection assay. The binding of K8 to this region was confirmed in cells by using a chromatin immunoprecipitation method. Further analysis revealed that K8 binds to an extended region, and the entire region is 100% conserved between two KSHV ori-Lyt's. K8 protein displays significant similarity to the Zta protein of Epstein-Barr virus (EBV), which is a known OBP of EBV. This notion, together with the ability of K8 to bind to the KSHV ori-Lyt, suggests that K8 may function as an OBP in KSHV.

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.

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 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

2004 ◽  
Vol 78 (5) ◽  
pp. 2609-2614 ◽  
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.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Induces a Strong Bend on Binding to Terminal Repeat DNA

2005 ◽  
Vol 79 (21) ◽  
pp. 13829-13836 ◽  
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.

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

2015 ◽  
Vol 89 (20) ◽  
pp. 10206-10218 ◽  
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.

scholarly journals Identification of a virus trans-acting regulatory element on the latent DNA replication of Kaposi's sarcoma-associated herpesvirus

2004 ◽  
Vol 85 (4) ◽  
pp. 843-855 ◽  
Author(s):  
Chunghun Lim ◽  
Taegun Seo ◽  
Jun Jung ◽  
Joonho Choe
Keyword(s):  
Dna Replication ◽  
Cell Lines ◽  
Kaposi's Sarcoma ◽  
Regulatory Element ◽  
Kaposi’S Sarcoma ◽  
Virus Genome ◽  
Nuclear Antigen ◽  
N Terminus ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays a pivotal role in the maintenance of the virus genome in latently infected cells. LANA1 links virus genomes to host chromosomes via a C-terminal DNA-binding domain which interacts with the sequences located in terminal repeats (TRs) of the virus genome and via an N-terminal chromosome-binding sequence which associates with the host chromosomes, respectively. Recent data suggest that LANA1 also actively participates in the replication of KSHV TR-containing plasmid in the transient DNA replication assay. In this report, it was found that C33A and COS-1, but not NIH/3T3, cell lines are permissive for the transient replication of KSHV TR-containing plasmid. Using several LANA1-deletion mutants, the minimum domain of LANA1 required for replication activity was also determined. In addition, the N terminus of LANA1 inhibited the transient replication systems of KSHV and Epstein–Barr virus (EBV) in transiently transfected 293 and 293T cells, but the C terminus of LANA1 specifically inhibited the transient replication system of KSHV in other cell lines. Consistent with previous reports, these data further emphasize the functional importance of the N terminus of LANA1 on replication from the KSHV latent origin of DNA replication.

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