scholarly journals Functional Characterization of Kaposi's Sarcoma-Associated Herpesvirus ORF45 byBacterial Artificial Chromosome-Based Mutagenesis

2006 ◽  
Vol 80 (24) ◽  
pp. 12187-12196 ◽  
Author(s):  
Fan Xiu Zhu ◽  
Xiaojuan Li ◽  
Fuchun Zhou ◽  
Shou-Jiang Gao ◽  
Yan Yuan
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Artificial Chromosome ◽  
Viral Gene ◽  
Type I ◽  
Wild Type ◽  
Early Protein ◽  
Significant Difference ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an immediate-early protein. This protein is also present in virions as a tegument protein. ORF45 protein interacts with interferon regulatory factor 7 (IRF-7) and inhibits virus-induced type I interferon production by blocking activation of IRF-7. To define further the function of ORF45 and the mechanism underlying its action, we constructed an ORF45-null recombinant virus genome (BAC-stop45) by using a bacterial artificial chromosome (BAC) system. Stable 293T cells carrying the BAC36 (wild type) and BAC-stop45 genomes were generated. When monolayers of 293T BAC36 and 293T BAC-stop45 cells were induced with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate, no significant difference was found between them in overall viral gene expression and lytic DNA replication, but induced 293T BAC-stop45 cells released 10-fold fewer virions to the medium than did 293T BAC36 cells. When ORF45-null virus was used to infect cells, lower infectivity was observed than for wild-type BAC36. These results suggest that KSHV ORF45 plays roles in both early and late stages of viral infection, probably in viral ingress and egress.

scholarly journals Construction of an Infectious Rhesus Rhadinovirus Bacterial Artificial Chromosome for the Analysis of Kaposi's Sarcoma-Associated Herpesvirus-Related Disease Development

2007 ◽  
Vol 81 (6) ◽  
pp. 2957-2969 ◽  
Author(s):  
Ryan D. Estep ◽  
Michael F. Powers ◽  
Bonnie K. Yen ◽  
He Li ◽  
Scott W. Wong
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Artificial Chromosome ◽  
Wild Type ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Rhesus rhadinovirus (RRV) is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) and causes KSHV-like diseases in immunocompromised rhesus macaques (RM) that resemble KSHV-associated diseases including multicentric Castleman's disease and non-Hodgkin's lymphoma. RRV retains a majority of open reading frames (ORFs) postulated to be involved in the pathogenesis of KSHV and is the closest available animal model to KSHV infection in humans. Here we describe the generation of a recombinant clone of RRV strain 17577 (RRV17577) utilizing bacterial artificial chromosome (BAC) technology. Characterization of the RRV BAC demonstrated that it is a pathogenic molecular clone of RRV17577, producing virus that behaves like wild-type RRV both in vitro and in vivo. Specifically, BAC-derived RRV displays wild-type growth properties in vitro and readily infects simian immunodeficiency virus-infected RM, inducing B cell hyperplasia, persistent lymphadenopathy, and persistent infection in these animals. This RRV BAC will allow for rapid genetic manipulation of the RRV genome, facilitating the creation of recombinant versions of RRV that harbor specific alterations and/or deletions of viral ORFs. This system will provide insights into the roles of specific RRV genes in various aspects of the viral life cycle and the RRV-associated pathogenesis in vivo in an RM model of infection. Furthermore, the generation of chimeric versions of RRV containing KSHV genes will allow analysis of the function and contributions of KSHV genes to viral pathogenesis by using a relevant primate model system.

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 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 Sensitivity of the C-Terminal Nuclease Domain of Kaposi's Sarcoma-Associated Herpesvirus ORF29 to Two Classes of Active-Site Ligands

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Jennifer T. Miller ◽  
Haiyan Zhao ◽  
Takashi Masaoka ◽  
Brittany Varnado ◽  
Elena M. Cornejo Castro ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Active Site ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Nuclease Activity ◽  
Divalent Metal ◽  
Wild Type ◽  
Nuclease Domain ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma, belongs to theHerpesviridaefamily, whose members employ a multicomponent terminase to resolve nonparametric viral DNA into genome-length units prior to their packaging. Homology modeling of the ORF29 C-terminal nuclease domain (pORF29C) and bacteriophage Sf6 gp2 have suggested an active site clustered with four acidic residues, D476, E550, D661, and D662, that collectively sequester the catalytic divalent metal (Mn2+) and also provided important insight into a potential inhibitor binding mode. Using this model, we have expressed, purified, and characterized the wild-type pORF29C and variants with substitutions at the proposed active-site residues. Differential scanning calorimetry demonstrated divalent metal-induced stabilization of wild-type (WT) and D661A pORF29C, consistent with which these two enzymes exhibited Mn2+-dependent nuclease activity, although the latter mutant was significantly impaired. Thermal stability of WT and D661A pORF29C was also enhanced by binding of an α-hydroxytropolone (α-HT) inhibitor shown to replace divalent metal at the active site. For the remaining mutants, thermal stability was unaffected by divalent metal or α-HT binding, supporting their role in catalysis. pORF29C nuclease activity was also inhibited by two classes of small molecules reported to inhibit HIV RNase H and integrase, both of which belong to the superfamily of nucleotidyltransferases. Finally, α-HT inhibition of KSHV replication suggests ORF29 nuclease function as an antiviral target that could be combined with latency-activating compounds as a shock-and-kill antiviral strategy.

scholarly journals Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus duringDe NovoPrimary Infection of Human B and Endothelial Cells

2014 ◽  
Vol 89 (6) ◽  
pp. 3093-3111 ◽  
Author(s):  
Pravinkumar Purushothaman ◽  
Suhani Thakker ◽  
Subhash C. Verma
Keyword(s):  
Primary Infection ◽  
Kaposi's Sarcoma ◽  
De Novo ◽  
Kaposi’S Sarcoma ◽  
Viral Gene ◽  
Target Cells ◽  
Viral Genes ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTKaposi's sarcoma-associated herpesvirus (KSHV) infects many target cells (e.g., endothelial, epithelial, and B cells, keratinocytes, and monocytes) to establish lifelong latent infections. Viral latent-protein expression is critical in inducing and maintaining KSHV latency. Infected cells are programmed to retain the incoming viral genomes during primary infection. Immediately after infection, KSHV transcribes many lytic genes that modulate various cellular pathways to establish successful infection. Analysis of the virion particle showed that the virions contain viral mRNAs, microRNAs, and other noncoding RNAs that are transduced into the target cells during infection, but their biological functions are largely unknown. We performed a comprehensive analysis of the KSHV virion packaged transcripts and the profiles of viral genes transcribed afterde novoinfections of various cell types (human peripheral blood mononuclear cells [PBMCs], CD14+monocytes, and telomerase-immortalized vascular endothelial [TIVE] cells), from viral entry until latency establishment. A next-generation sequence analysis of the total transcriptome showed that several viral RNAs (polyadenylated nuclear RNA, open reading frame 58 [ORF58], ORF59, T0.7, and ORF17) were abundantly present in the KSHV virions and effectively transduced into the target cells. Analysis of the transcription profiles of each viral gene showed specific expression patterns in different cell lines, with the majority of the genes, other than latent genes, silencing after 24 h postinfection. We differentiated the actively transcribing genes from the virion-transduced transcripts using a nascent RNA capture approach (Click-iT chemistry), which identified transcription of a number of viral genes during primary infection. Treating the infected cells with phosphonoacetic acid (PAA) to block the activity of viral DNA polymerase confirmed the involvement of lytic DNA replication during primary infection. To further understand the role of DNA replication during primary infection, we performedde novoPBMC infections with a recombinant ORF59-deleted KSHV virus, which showed significantly reduced numbers of viral copies in the latently infected cells. In summary, the transduced KSHV RNAs as well as the actively transcribed genes control critical processes of early infection to establish KSHV latency.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of multiple human malignancies in immunocompromised individuals. KSHV establishes a lifelong latency in the infected host, during which only a limited number of viral genes are expressed. However, a fraction of latently infected cells undergo spontaneous reactivation to produce virions that infect the surrounding cells. These newly infected cells are primed early to retain the incoming viral genome and induce cell growth. KSHV transcribes a variety of lytic proteins duringde novoinfections that modulate various cellular pathways to establish the latent infection. Interestingly, a large number of viral proteins and RNA are encapsidated in the infectious virions and transduced into the infected cells during ade novoinfection. This study determined the kinetics of the viral gene expression duringde novoKSHV infections and the functional role of the incoming viral transcripts in establishing latency.

scholarly journals ORF33 and ORF38 of Kaposi's Sarcoma-Associated Herpesvirus Interact and Are Required for Optimal Production of Infectious Progeny Viruses

2015 ◽  
Vol 90 (4) ◽  
pp. 1741-1756 ◽  
Author(s):  
Jian-jun Wu ◽  
Denis Avey ◽  
Wenwei Li ◽  
Joseph Gillen ◽  
Bishi Fu ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Viral Gene ◽  
Virus Propagation ◽  
Progeny Virion ◽  
Cytoplasmic Vesicles ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTWe recently showed that the interaction between Kaposi's sarcoma-associated herpesvirus (KSHV) tegument proteins ORF33 and ORF45 is crucial for progeny virion production, but the exact functions of KSHV ORF33 during lytic replication were unknown (J. Gillen, W. Li, Q. Liang, D. Avey, J. Wu, F. Wu, J. Myoung, and F. Zhu, J Virol89:4918–4931, 2015,http://dx.doi.org/10.1128/JVI.02925-14). Therefore, here we investigated the relationship between ORF33 and ORF38, whose counterparts in both alpha- and betaherpesviruses interact with each other. Using specific monoclonal antibodies, we found that both proteins are expressed during the late lytic cycle with similar kinetics and that both are present in mature virions as components of the tegument. Furthermore, we confirmed that ORF33 interacts with ORF38. Interestingly, we observed that ORF33 tightly associates with the capsid, whereas ORF38 associates with the envelope. We generated ORF33-null, ORF38-null, and double-null mutants and found that these mutants apparently have identical phenotypes: the mutations caused no apparent effect on viral gene expression but reduced the yield of progeny virion by about 10-fold. The progeny virions also lack certain virion component proteins, including ORF45. During viral lytic replication, the virions associate with cytoplasmic vesicles. We also observed that ORF38 associates with the membranes of vesicles and colocalizes with the Golgi membrane or early endosome membrane. Further analyses of ORF33/ORF38 mutants revealed the reduced production of virion-containing vesicles, suggesting that ORF33 and ORF38 are involved in the transport of newly assembled viral particles into cytoplasmic vesicles, a process important for viral maturation and egress.IMPORTANCEHerpesvirus assembly is an essential step in virus propagation that leads to the generation of progeny virions. It is a complicated process that depends on the delicate regulation of interactions among virion proteins. We previously revealed an essential role of ORF45-ORF33 binding for virus assembly. Here, we report that ORF33 and its binding partner, ORF38, are required for infectious virus production due to their important role in the tegumentation process. Moreover, we found that both ORF33 and ORF38 are involved in the transportation of virions through vesicles during maturation and egress. Our results provide new insights into the important roles of ORF33 and ORF38 during viral assembly, a process critical for virus propagation that is intimately linked to KSHV pathobiology.

scholarly journals Disintegrin-like domain of glycoprotein B regulates Kaposi’s sarcoma-associated herpesvirus infection of cells

2014 ◽  
Vol 95 (8) ◽  
pp. 1770-1782 ◽  
Author(s):  
Lia R. Walker ◽  
Hosni A. M. Hussein ◽  
Shaw M. Akula
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Glycoprotein B ◽  
Binding Motif ◽  
Wild Type ◽  
Infection Rates ◽  
Herpesvirus Infection ◽  
Kshv Infection ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) glycoprotein B (gB) is a lytic structural protein expressed on the envelope of mature virions and on the membrane of cells supporting lytic infection. In addition to this viral glycoprotein’s interaction with integrins via its RGD (Arg-Gly-Asp) motif, KSHV gB possesses a disintegrin-like domain (DLD), which binds integrins as well. Prior to this study, there has been minimal research involving the less common integrin-binding motif, DLD, of gB as it pertains to herpesvirus infection. By using phage display peptide library screening and molecular biology techniques, the DLD of KSHV gB was shown to interact specifically with non-RGD binding α9β1 integrins. Similarly, monitoring wild-type infection confirmed α9β1:DLD interactions to be critical to successful KSHV infection of human foreskin fibroblast (HFF) cells and human dermal microvascular endothelial cells (HMVEC-d) compared with 293 cells. To further demonstrate the importance of the DLD of gB in KSHV infection, two recombinant virus constructs were generated using a bacterial artificial chromosome (BAC) system harbouring the KSHV genome (BAC36): BAC36ΔD-KSHV (lacking a functionally intact DLD of gB and containing an introduced tetracycline cassette) and BAC36.T-KSHV (containing an intact DLD sequence and an introduced tetracycline cassette). Accordingly, BAC36ΔD-KSHV presented significantly lower infection rates in HFF and HMVEC-d cells compared with the comparable infection rates achieved by wild-type BAC36-KSHV and BAC36.T-KSHV. Thus, the present report has delineated a critical role for the DLD of gB in KSHV infection, which may lead to a broader knowledge regarding the sophisticated mechanisms utilized by virus-encoded structural proteins in KSHV entry and infection.

scholarly journals Epigenetic Regulation of Kaposi's Sarcoma-Associated Herpesvirus Latency by Virus-Encoded MicroRNAs That Target Rta and the Cellular Rbl2-DNMT Pathway

2010 ◽  
Vol 84 (6) ◽  
pp. 2697-2706 ◽  
Author(s):  
Fang Lu ◽  
William Stedman ◽  
Malik Yousef ◽  
Rolf Renne ◽  
Paul M. Lieberman
Keyword(s):  
Kaposi's Sarcoma ◽  
Histone H3 ◽  
Indirect Comparison ◽  
Kaposi’S Sarcoma ◽  
Immediate Early ◽  
Mrna Levels ◽  
Sequence Alignments ◽  
Early Protein ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a cluster of 12 microRNAs (miRNAs) that are processed from a transcript that is embedded within the major latency control region. We have generated a deletion mutation that eliminates 10 of the 12 viral miRNAs from the KSHV bacmid by using recombineering methods. The KSHV miRNA deletion mutant (BAC36 ΔmiR) behaved similarly to wild-type (wt) BAC36 in viral production, latency gene transcription, and viral DNA copy number in 293 and dermal microvascular endothelial cells (DMVECs). However, BAC36 ΔmiR consistently expressed elevated levels of viral lytic genes, including the immediate-early transcriptional activator Rta (ORF50). At least one KSHV microRNA (miRK12-5) was capable of suppressing ORF50 mRNA, but poor seed sequence alignments suggest that these targets may be indirect. Comparison of epigenetic marks in ΔmiR KSHV genomes revealed decreases in histone H3 K9 methylation, increases in histone H3 acetylation, and a striking loss of DNA methylation throughout the viral and cellular genome. One viral miRNA, K12-4-5p, was found to have a sequence targeting retinoblastoma (Rb)-like protein 2 (Rbl2), which is a known repressor of DNA methyl transferase 3a and 3b mRNA transcription. We show that ectopic expression of miR-K12-4-5p reduces Rbl2 protein expression and increases DNMT1, -3a, and -3b mRNA levels relative to the levels for control cells. We conclude that KSHV miRNA targets multiple pathways to maintain the latent state of the KSHV genome, including repression of the viral immediate-early protein Rta and a cellular factor, Rbl2, that regulates global epigenetic reprogramming.

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