scholarly journals Kaposi’s sarcoma-associated herpesvirus (KSHV) processivity factor, ORF59, binds to canonical and linker histones, and carboxy-terminus is dispensable for viral DNA synthesis

2020 ◽  
Author(s):  
Isaura Vanessa Gutierrez ◽  
Poulomee Sarkar ◽  
Cyprian C. Rossetto
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Nuclear Localization ◽  
Kaposi's Sarcoma ◽  
Essential Role ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Oncogenic Virus ◽  
Viral Dna ◽  
Processivity Factor

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus and the causative agent of Kaposi’s sarcoma, multicentric Castleman’s disease, and primary effusion lymphoma. During lytic reactivation there is a temporal cascade of viral gene expression that results in the production of new virions. One of the viral factors that is expressed during reactivation is open reading frame (ORF) 59, the viral DNA polymerase processivity factor. ORF59 plays an essential role for DNA synthesis and is required for the nuclear localization of the viral DNA polymerase (ORF9) to the origin of lytic replication (oriLyt). In addition to its functions in viral DNA synthesis, ORF59 has been shown to interact with chromatin complexes including histones and cellular methyltransferases. In this study, a series of KSHV BACmids containing 50 aa deletions within ORF59 were generated to determine the interaction domains between ORF59 and histones, as well as to assess the effects on replication fitness as a result of these interactions. These studies show that in the context of infection ORF59 51-100 and 151-200 amino acids (aa) are required for interaction with histones, and ORF59 301-396 aa are not required for DNA synthesis. Since full length ORF59 is known to localize to the nucleus, we performed immunofluorescent assay (IFA) with the ORF59 deletion mutants and showed that all deletions are localized to the nucleus, this includes the ORF59 deletion without the previously identified nuclear localization signal (NLS). These studies further characterize ORF59 and demonstrate its essential role during lytic replication. IMPORTANCE. KSHV is an oncogenic virus and the causative agent of potentially fatal malignancies. Lytic replication of KSHV is an essential part of the viral lifecycle, allowing for virus dissemination within the infected host and shedding to infect naïve hosts. Viral DNA synthesis is a critical step in the production of new infectious virions. One of the proteins that is vital to this process is ORF59, the viral encoded polymerase processivity factor. Previous work has demonstrated that the function of ORF59 is closely connected to its association with other viral and cellular factors. The studies presented here extend that work to include the interaction between ORF59 and histones. This interaction offers an additional level of regulation of the chromatinized viral genome, ultimately influencing DNA synthesis and transcription dynamics.

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

2008 ◽  
Vol 83 (6) ◽  
pp. 2531-2539 ◽  
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.

scholarly journals Nucleolin Associates with the Human Cytomegalovirus DNA Polymerase Accessory Subunit UL44 and Is Necessary for Efficient Viral Replication

2009 ◽  
Vol 84 (4) ◽  
pp. 1771-1784 ◽  
Author(s):  
Blair L. Strang ◽  
Steeve Boulant ◽  
Donald M. Coen
Keyword(s):  
Infected Cell ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Western Blotting ◽  
Human Cytomegalovirus ◽  
Major Protein ◽  
Cell Nuclei ◽  
Viral Dna ◽  
Infected Cells ◽  
Processivity Factor

ABSTRACT In the eukaryotic cell, DNA replication entails the interaction of multiple proteins with the DNA polymerase processivity factor PCNA. As the structure of the presumptive human cytomegalovirus (HCMV) DNA polymerase processivity factor UL44 is highly homologous to that of PCNA, we hypothesized that UL44 also interacts with numerous proteins. To investigate this possibility, recombinant HCMV expressing FLAG-tagged UL44 was generated and used to immunoprecipitate UL44 and associated proteins from infected cell lysates. Unexpectedly, nucleolin, a major protein component of the nucleolus, was identified among these proteins by mass spectrometry and Western blotting. The association of nucleolin and UL44 in infected cell lysate was confirmed by reciprocal coimmunoprecipitation in the presence and absence of nuclease. Western blotting and immunofluorescence assays demonstrated that the level of nucleolin increases during infection and that nucleolin becomes distributed throughout the nucleus. Furthermore, the colocalization of nucleolin and UL44 in infected cell nuclei was observed by immunofluorescence assays. Assays of HCMV-infected cells treated with small interfering RNA (siRNA) targeting nucleolin mRNA indicated that nucleolin was required for efficient virus production, viral DNA synthesis, and the expression of a late viral protein, with a correlation between the efficacy of knockdown and the effect on virus replication. In contrast, the level of neither global protein synthesis nor the replication of an unrelated virus (reovirus) was reduced in siRNA-treated cells. Taken together, our results indicate an association of nucleolin and UL44 in HCMV-infected cells and a role for nucleolin in viral DNA synthesis.

scholarly journals Identification of the Essential Role of Viral Bcl-2 for Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication

2015 ◽  
Vol 89 (10) ◽  
pp. 5308-5317 ◽  
Author(s):  
Qiming Liang ◽  
Brian Chang ◽  
Patrick Lee ◽  
Kevin F. Brulois ◽  
Jianning Ge ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Dramatic Reduction ◽  
Viral Dna ◽  
Lytic Gene ◽  
Lytic Gene Expression ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) evades host defenses through tight suppression of autophagy by targeting each step of its signal transduction: by viral Bcl-2 (vBcl-2) in vesicle nucleation, by viral FLIP (vFLIP) in vesicle elongation, and by K7 in vesicle maturation. By exploring the roles of KSHV autophagy-modulating genes, we found, surprisingly, that vBcl-2 is essential for KSHV lytic replication, whereas vFLIP and K7 are dispensable. Knocking out vBcl-2 from the KSHV genome resulted in decreased lytic gene expression at the mRNA and protein levels, a lower viral DNA copy number, and, consequently, a dramatic reduction in the amount of progeny infectious viruses, as also described in the accompanying article (A. Gelgor, I. Kalt, S. Bergson, K. F. Brulois, J. U. Jung, and R. Sarid, J Virol 89:5298–5307, 2015). More importantly, the antiapoptotic and antiautophagic functions of vBcl-2 were not required for KSHV lytic replication. Using a comprehensive mutagenesis analysis, we identified that glutamic acid 14 (E14) of vBcl-2 is critical for KSHV lytic replication. Mutating E14to alanine totally blocked KSHV lytic replication but showed little or no effect on the antiapoptotic and antiautophagic functions of vBcl-2. Our study indicates that vBcl-2 harbors at least three important and genetically separable functions to modulate both cellular signaling and the virus life cycle.IMPORTANCEThe present study shows for the first time that vBcl-2 is essential for KSHV lytic replication. Removal of the vBcl-2 gene results in a lower level of KSHV lytic gene expression, impaired viral DNA replication, and consequently, a dramatic reduction in the level of progeny production. More importantly, the role of vBcl-2 in KSHV lytic replication is genetically separated from its antiapoptotic and antiautophagic functions, suggesting that the KSHV Bcl-2 carries a novel function in viral lytic replication.

scholarly journals Localization of Double-Strand Break Repair Proteins to Viral Replication Compartments following Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus

2017 ◽  
Vol 91 (22) ◽  
Author(s):  
Robert Hollingworth ◽  
Richard D. Horniblow ◽  
Calum Forrest ◽  
Grant S. Stewart ◽  
Roger J. Grand
Keyword(s):  
Dna Damage ◽  
Viral Replication ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Viral Dna ◽  
Dsb Repair ◽  
Viral Genomes ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Double-strand breaks (DSBs) in DNA are recognized by the Ku70/80 heterodimer and the MRE11-RAD50-NBS1 (MRN) complex and result in activation of the DNA-PK and ATM kinases, which play key roles in regulating the cellular DNA damage response (DDR). DNA tumor viruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) are known to interact extensively with the DDR during the course of their replicative cycles. Here we show that during lytic amplification of KSHV DNA, the Ku70/80 heterodimer and the MRN complex consistently colocalize with viral genomes in replication compartments (RCs), whereas other DSB repair proteins form foci outside RCs. Depletion of MRE11 and abrogation of its exonuclease activity negatively impact viral replication, while in contrast, knockdown of Ku80 and inhibition of the DNA-PK enzyme, which are involved in nonhomologous end joining (NHEJ) repair, enhance amplification of viral DNA. Although the recruitment of DSB-sensing proteins to KSHV RCs is a consistent occurrence across multiple cell types, activation of the ATM-CHK2 pathway during viral replication is a cell line-specific event, indicating that recognition of viral DNA by the DDR does not necessarily result in activation of downstream signaling pathways. We have also observed that newly replicated viral DNA is not associated with cellular histones. Since the presence and modification of these DNA-packaging proteins provide a scaffold for docking of multiple DNA repair factors, the absence of histone deposition may allow the virus to evade localization of DSB repair proteins that would otherwise have a detrimental effect on viral replication. IMPORTANCE Tumor viruses are known to interact with machinery responsible for detection and repair of double-strand breaks (DSBs) in DNA, although detail concerning how Kaposi's sarcoma-associated herpesvirus (KSHV) modulates these cellular pathways during its lytic replication phase was previously lacking. By undertaking a comprehensive assessment of the localization of DSB repair proteins during KSHV replication, we have determined that a DNA damage response (DDR) is directed to viral genomes but is distinct from the response to cellular DNA damage. We also demonstrate that although recruitment of the MRE11-RAD50-NBS1 (MRN) DSB-sensing complex to viral genomes and activation of the ATM kinase can promote KSHV replication, proteins involved in nonhomologous end joining (NHEJ) repair restrict amplification of viral DNA. Overall, this study extends our understanding of the virus-host interactions that occur during lytic replication of KSHV and provides a deeper insight into how the DDR is manipulated during viral infection.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus mRNA Accumulation in Nuclear Foci Is Influenced by Viral DNA Replication and Viral Noncoding Polyadenylated Nuclear RNA

2018 ◽  
Vol 92 (13) ◽  
Author(s):  
Tenaya K. Vallery ◽  
Johanna B. Withers ◽  
Joana A. Andoh ◽  
Joan A. Steitz
Keyword(s):  
Dna Replication ◽  
Viral Replication ◽  
Dna Synthesis ◽  
Host Cell ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV), like other herpesviruses, replicates within the nuclei of its human cell host and hijacks host machinery for expression of its genes. The activities that culminate in viral DNA synthesis and assembly of viral proteins into capsids physically concentrate in nuclear areas termed viral replication compartments. We sought to better understand the spatiotemporal regulation of viral RNAs during the KSHV lytic phase by examining and quantifying the subcellular localization of select viral transcripts. We found that viral mRNAs, as expected, localized to the cytoplasm throughout the lytic phase. However, dependent on active viral DNA replication, viral transcripts also accumulated in the nucleus, often in foci in and around replication compartments, independent of the host shutoff effect. Our data point to involvement of the viral long noncoding polyadenylated nuclear (PAN) RNA in the localization of an early, intronless viral mRNA encoding ORF59-58 to nuclear foci that are associated with replication compartments.IMPORTANCELate in the lytic phase, mRNAs from Kaposi's sarcoma-associated herpesvirus accumulate in the host cell nucleus near viral replication compartments, centers of viral DNA synthesis and virion production. This work contributes spatiotemporal data on herpesviral mRNAs within the lytic host cell and suggests a mechanism for viral RNA accumulation. Our findings indicate that the mechanism is independent of the host shutoff effect and splicing but dependent on active viral DNA synthesis and in part on the viral noncoding RNA, PAN RNA. PAN RNA is essential for the viral life cycle, and its contribution to the nuclear accumulation of viral messages may facilitate propagation of the virus.

scholarly journals Kaposi’s sarcoma-associated herpesvirus processivity factor (PF-8) recruits cellular E3 ubiquitin ligase CHFR to promote PARP1 degradation and lytic replication

2021 ◽  
Vol 17 (1) ◽  
pp. e1009261
Author(s):  
Woo-Chang Chung ◽  
Seungrae Lee ◽  
Yejin Kim ◽  
Jong Bok Seo ◽  
Moon Jung Song
Keyword(s):  
Kaposi's Sarcoma ◽  
Direct Interaction ◽  
Kaposi’S Sarcoma ◽  
Ubiquitin Proteasome System ◽  
Inhibitory Effect ◽  
Lytic Replication ◽  
Transactivation Activity ◽  
Processivity Factor ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Kaposi’s sarcoma–associated herpesvirus (KSHV), which belongs to the gammaherpesvirus subfamily, is associated with the pathogenesis of various tumors. Nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) catalyzes the polymerization of ADP-ribose units on target proteins. In KSHV-infected cells, PARP1 inhibits replication and transcription activator (RTA), a molecular switch that initiates lytic replication, through direct interaction. Thus, for efficient replication, KSHV has to overcome the molecular barrier in the form of PARP1. Previously, we have demonstrated that KSHV downregulates the expression of PARP1 through PF-8, a viral processivity factor. PF-8 induces ubiquitin–proteasome system–mediated degradation of PARP1 via direct physical association and enhances RTA transactivation activity. Here, we showed that dimerization domains of PF-8 are crucial not only for PARP1 interaction and degradation but also for enhancement of the RTA transactivation activity. PF-8 recruited CHFR for the PARP1 degradation. A knockdown of CHFR attenuated the PF-8–induced PARP1 degradation and enhancement of the RTA transactivation activity, leading to reduced KSHV lytic replication. These findings reveal a mechanism by which KSHV PF-8 recruits a cellular E3 ligase to curtail the inhibitory effect of PARP1 on KSHV lytic replication.

scholarly journals RNA Editing of the Human Herpesvirus 8 Kaposin Transcript Eliminates Its Transforming Activity and Is Induced during Lytic Replication

2007 ◽  
Vol 81 (24) ◽  
pp. 13544-13551 ◽  
Author(s):  
Sharon Z. Gandy ◽  
Sarah D. Linnstaedt ◽  
Sumitra Muralidhar ◽  
Kathleen A. Cashman ◽  
Leonard J. Rosenthal ◽  
...  
Keyword(s):  
Rna Editing ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Human Herpesvirus 8 ◽  
Viral Dna ◽  
Herpesvirus 8 ◽  
Sequence Heterogeneity ◽  
Transforming Activity

ABSTRACT Human herpesvirus 8 is the etiologic agent associated with Kaposi's sarcoma and primary effusion lymphoma (PEL). The K12 RNA, which produces as many as three variants of the kaposin protein, as well as a microRNA, is the most abundant transcript expressed in latent Kaposi's sarcoma-associated herpesvirus infection, and yet it is also induced during lytic replication. The portion of the transcript that includes the microRNA and the kaposin A sequence has been shown to have tumorigenic potential. Genome coordinate 117990, which is within this transcript, has been found to be heterogeneous, primarily in RNAs but also among viral DNA sequences. This sequence heterogeneity affects an amino acid in kaposins A and C and the microRNA. The functional effects of this sequence heterogeneity have not been studied, and its origin has not been definitively settled; both RNA editing and heterogeneity at the level of the viral genome have been proposed. Here, we show that transcripts containing A at position 117990 are tumorigenic, while those with G at this position are not. Using a highly sensitive quantitative assay, we observed that, in PEL cells under conditions where more than 60% of cDNAs derived from K12 RNA transcripts have G at coordinate 117990, there is no detectable G in the viral DNA sequence at this position, only A. This result is consistent with RNA editing by one of the host RNA adenosine deaminases (ADARs). Indeed, we observed that purified human ADAR1 efficiently edits K12 RNA in vitro. Remarkably, the amount of editing correlated with the replicative state of the virus; editing levels were nearly 10-fold higher in cells treated to induce lytic viral replication. These results suggest that RNA editing controls the function of one segment of the kaposin transcript, such that it has transforming activity during latent replication and possibly another, as-yet-undetermined, function during lytic replication.

scholarly journals Cloning and Functional Analysis of Kaposi’s Sarcoma-Associated Herpesvirus DNA Polymerase and Its Processivity Factor

1998 ◽  
Vol 72 (7) ◽  
pp. 6228-6232 ◽  
Author(s):  
Kai Lin ◽  
Charlotte Y. Dai ◽  
Robert P. Ricciardi
Keyword(s):  
Dna Polymerase ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Herpes Simplex Virus 1 ◽  
Processivity Factor ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Simplex Virus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is a newly identified virus with tumorigenic potential. Here, we cloned and expressed the DNA polymerase (Pol-8) of KSHV and its processivity factor (PF-8). Pol-8 bound specifically to PF-8 in vitro. Moreover, the DNA synthesis activity of Pol-8 was shown in vitro to be strongly dependent on PF-8. Addition of PF-8 to Pol-8 allowed efficient synthesis of fully extended DNA products corresponding to the full-length M13 template (7,249 nucleotides), whereas Pol-8 alone could incorporate only several nucleotides. The specificity of PF-8 and Pol-8 for each other was demonstrated by their inability to be functionally replaced by the DNA polymerases and processivity factors of herpes simplex virus 1 and human herpesvirus 6.

scholarly journals The Late Promoter of the Human Cytomegalovirus Viral DNA Polymerase Processivity Factor Has an Impact on Delayed Early and Late Viral Gene Products but Not on Viral DNA Synthesis

2007 ◽  
Vol 81 (12) ◽  
pp. 6197-6206 ◽  
Author(s):  
Hiroki Isomura ◽  
Mark F. Stinski ◽  
Ayumi Kudoh ◽  
Sanae Nakayama ◽  
Satoko Iwahori ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Wild Type ◽  
Viral Dna ◽  
Recombinant Viruses ◽  
Tata Element ◽  
Processivity Factor

ABSTRACT Transcription of the DNA polymerase processivity factor gene (UL44) of human cytomegalovirus initiates at three distinct start sites, which are differentially regulated during productive infection. Two of these start sites, the distal and proximal sites, are active at early times, and the middle start site is active at only late times after infection (F. Leach and E. S. Mocarski, J. Virol. 63:1783-1791, 1989). Compared to the wild type, UL44 gene expression was lower for recombinant viruses with the distal or the middle TATA element mutated. The transcripts initiating from the distal or middle start site facilitated late viral gene expression. The level of viral DNA synthesis was affected by mutation of the distal TATA element. In contrast, mutation of the middle TATA element did not affect the level of viral DNA synthesis, but it did affect significantly the level of late viral gene expression. Recombinant viruses with the distal or middle TATA element mutated grew more slowly than the wild type at both low and high multiplicities of infection. Reduced expression of the UL44 gene from the late middle viral promoter correlated with decreased late viral protein expression and decreased viral growth.

Sign in / Sign up

Export Citation Format

Share Document