scholarly journals The ORF45 Protein of Kaposi's Sarcoma-Associated Herpesvirus Is Associated with Purified Virions

2003 ◽  
Vol 77 (7) ◽  
pp. 4221-4230 ◽  
Author(s):  
Fan Xiu Zhu ◽  
Yan Yuan
Keyword(s):  
Kaposi's Sarcoma ◽  
Early Stage ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kaposi’S Sarcoma ◽  
Viral Envelope ◽  
Host Cells ◽  
Early Gene ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) ORF45 is encoded by an immediate-early gene in the KSHV genome. This protein was recently shown to interact with interferon regulatory factor 7 and inhibit virus-mediated alpha/beta interferon induction (Zhu et al., Proc. Natl. Acad. Sci. USA 99:5573-5578, 2002). ORF45 was characterized as a phosphorylated protein, and it is localized in the cytoplasm of infected cells. In this report, we provide evidence that ORF45 is associated with KSHV virions. (i) ORF45 was detected in gradient-purified virions by Western blotting along with known structural proteins of KSHV including gB, K8.1, and major capsid protein. In contrast, ORF50/Rta, K8α, and ORF59/PF8 were not detected in the same virion preparation. (ii) ORF45 comigrates with KSHV virions in sucrose gradient ultracentrifugation. (iii) Virion-associated ORF45 was resistant to trypsin digestion but became sensitive after the virions were treated with detergent which destroys the viral envelope. (iv) ORF45 remained associated with tegument-nucleocapsid complex when virion-specific glycoproteins were removed after detergent treatment. (v) An ORF45 protein band was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extensively purified KSHV virions and identified by mass spectrometry. (vi) By immunoelectron microscopy, virus-like structures were specifically stained by anti-ORF45 antibody. Based on the evidence, we conclude that ORF45 is associated with purified KSHV virions and appears to be a tegument protein. The presence of ORF45 in KSHV virions raised the possibility that this protein may be delivered to host cells at the start of infection and therefore have the opportunity to act at the very early stage of the infection, suggesting an important role of ORF45 in KSHV primary infection.

scholarly journals Identification of the Novel K15 Gene at the Rightmost End of the Kaposi's Sarcoma-Associated Herpesvirus Genome

2000 ◽  
Vol 74 (1) ◽  
pp. 436-446 ◽  
Author(s):  
Joong-Kook Choi ◽  
Bok-Soo Lee ◽  
Sung N. Shim ◽  
Mengtao Li ◽  
Jae U. Jung
Keyword(s):  
Kaposi's Sarcoma ◽  
Biochemical Study ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kaposi’S Sarcoma ◽  
Binding Motif ◽  
Cytoplasmic Region ◽  
Splicing Pattern ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a distinct open reading frame called K15 at a position equivalent to the gene encoding LMP2A of Epstein-Barr virus (EBV). K15 isolates from body cavity-based lymphoma (BCBL) cells exhibited a dramatic sequence variation and a complex splicing pattern. However, all K15 alleles are organized similarly with the potential SH2 and SH3 binding motifs in their cytoplasmic regions. Northern blot analysis showed that K15 was weakly expressed in latently infected BCBL-1 cells, and the level of its expression was significantly induced by tetradecanoyl phorbol acetate stimulation. K15 encoded 40- to 55-kDa proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was localized at the cytoplasm and plasma membrane. To demonstrate the signal-transducing activity of the K15 protein, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8α polypeptide was replaced with that of KSHV K15. While the CD8-K15 chimera was not capable of eliciting cellular signal transduction upon stimulation with an anti-CD8 antibody, it significantly inhibited B-cell receptor signaling, as evidenced by a suppression of tyrosine phosphorylation and intracellular calcium mobilization. This inhibition required the putative SH2 or SH3 binding motif in the cytoplasmic region of K15. Biochemical study of CD8-K15 chimeras showed that the cytoplasmic region of K15 was constitutively tyrosine phosphorylated and that the tyrosine residue within the putative SH2 binding motif of K15 was a primary site of phosphorylation. These results demonstrate that KSHV K15 resembles LMP2A in genomic location, splicing pattern, and protein structure and by the presence of functional signal-transducing motifs in the cytoplasmic region. Thus, KSHV K15 is likely a distant evolutionary relative of EBV LMP2A.

scholarly journals Virion Proteins of Kaposi's Sarcoma-Associated Herpesvirus

2005 ◽  
Vol 79 (2) ◽  
pp. 800-811 ◽  
Author(s):  
Fan Xiu Zhu ◽  
Jae Min Chong ◽  
Lijun Wu ◽  
Yan Yuan
Keyword(s):  
Kaposi's Sarcoma ◽  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kaposi’S Sarcoma ◽  
Structure Identification ◽  
Functional Roles ◽  
Tegument Proteins ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The proteins that compose a herpesvirus virion are thought to contain the functional information required for de novo infection, as well as virion assembly and egress. To investigate functional roles of Kaposi's sarcoma-associated herpesvirus (KSHV) virion proteins in viral productive replication and de novo infection, we attempted to identify virion proteins from purified KSHV by a proteomic approach. Extracellular KSHV virions were purified from phorbol-12-tetradecanoate-13-acetate-induced BCBL-1 cells through double-gradient ultracentrifugation, and their component proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thirty prominent protein bands were excised and subjected to high-performance liquid chromatography ion trap mass spectrometric analysis. This study led to the identification of 24 virion-associated proteins. These include five capsid proteins, eight envelope glycoproteins, six tegument proteins, and five proteins whose locations in the virions have not yet been defined. Putative tegument proteins encoded by open reading frame 21 (ORF21), ORF33, and ORF45 were characterized and found to be resistant to protease digestion when purified virions were treated with trypsin, confirming that they are located within the virion particles. The ORF64-encoded large tegument protein was found to be associated with capsid but sensitive to protease treatment, suggesting its unique structure and array in KSHV virions. In addition, cellular β-actin and class II myosin heavy chain type A were found inside KSHV virions and associated with tegument-capsid structure. Identification of KSHV virion proteins makes it possible to study the functional roles of these virion proteins in KSHV replication and pathogenicity.

scholarly journals NF-κB Serves as a Cellular Sensor of Kaposi's Sarcoma-Associated Herpesvirus Latency and Negatively Regulates K-Rta by Antagonizing the RBP-Jκ Coactivator

2009 ◽  
Vol 83 (9) ◽  
pp. 4435-4446 ◽  
Author(s):  
Yoshihiro Izumiya ◽  
Chie Izumiya ◽  
Datsun Hsia ◽  
Thomas J. Ellison ◽  
Paul A. Luciw ◽  
...  
Keyword(s):  
Viral Protein ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Host Cells ◽  
Early Gene ◽  
Viral Gene ◽  
Viral Promoters ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Successful viral replication is dependent on a conducive cellular environment; thus, viruses must be sensitive to the state of their host cells. We examined the idea that an interplay between viral and cellular regulatory factors determines the switch from Kaposi's sarcoma-associated herpesvirus (KSHV) latency to lytic replication. The immediate-early gene product K-Rta is the first viral protein expressed and an essential factor in reactivation; accordingly, this viral protein is in a key position to serve as a viral sensor of cellular physiology. Our approach aimed to define a host transcription factor, i.e., host sensor, which modulates K-Rta activity on viral promoters. To this end, we developed a panel of reporter plasmids containing all 83 putative viral promoters for a comprehensive survey of the response to both K-Rta and cellular transcription factors. Interestingly, members of the NF-κB family were shown to be strong negative regulators of K-Rta transactivation for all but two viral promoters (Ori-RNA and K12). Recruitment of K-Rta to the ORF57 and K-bZIP promoters, but not the K12 promoter, was significantly impaired when NF-κB expression was induced. Many K-Rta-responsive promoters modulated by NF-κB contain the sequence of the RBP-Jκ binding site, a major coactivator which anchors K-Rta to target promoters via consensus motifs which overlap with that of NF-κB. Gel shift assays demonstrated that NF-κB inhibits the binding of RBP-Jκ and forms a complex with RBP-Jκ. Our results support a model in which a balance between K-Rta/RBP-Jκ and NF-κB activities determines KSHV reactivation. An important feature of this model is that the interplay between RBP-Jκ and NF-κB on viral promoters controls viral gene expression mediated by K-Rta.

scholarly journals Virus-Like Vesicles of Kaposi's Sarcoma-Associated Herpesvirus Activate Lytic Replication by Triggering Differentiation Signaling

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Danyang Gong ◽  
Xinghong Dai ◽  
Yuchen Xiao ◽  
Yushen Du ◽  
Travis J. Chapa ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Cell System ◽  
Lytic Replication ◽  
Host Cells ◽  
Mass Spectrometry Analysis ◽  
Productive Infection ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Virus-like vesicles (VLVs) are membrane-enclosed vesicles that resemble native enveloped viruses in organization but lack the viral capsid and genome. During the productive infection of tumor-associated gammaherpesviruses, both virions and VLVs are produced and are released into the extracellular space. However, studies of gammaherpesvirus-associated VLVs have been largely restricted by the technical difficulty of separating VLVs from mature virions. Here we report a strategy of selectively isolating VLVs by using a Kaposi's sarcoma-associated herpesvirus (KSHV) mutant that is defective in small capsid protein and is unable to produce mature virions. Using mass spectrometry analysis, we found that VLVs contained viral glycoproteins required for cellular entry, as well as tegument proteins involved in regulating lytic replication, but lacked capsid proteins. Functional analysis showed that VLVs induced the expression of the viral lytic activator RTA, initiating KSHV lytic gene expression. Furthermore, employing RNA sequencing, we performed a genomewide analysis of cellular responses triggered by VLVs and found that PRDM1, a master regulator in cell differentiation, was significantly upregulated. In the context of KSHV replication, we demonstrated that VLV-induced upregulation of PRDM1 was necessary and sufficient to reactivate KSHV by activating its RTA promoter. In sum, our study systematically examined the composition of VLVs and demonstrated their biological roles in manipulating host cell responses and facilitating KSHV lytic replication. IMPORTANCE Cells lytically infected with tumor-associated herpesviruses produce a high proportion of virus-like vesicles (VLVs). The composition and function of VLVs have not been well defined, largely due to the inability to efficiently isolate VLVs that are free of virions. Using a cell system capable of establishing latent KSHV infection and robust reactivation, we successfully isolated VLVs from a KSHV mutant defective in the small capsid protein. We quantitatively analyzed proteins and microRNAs in VLVs and characterized the roles of VLVs in manipulating host cells and facilitating viral infection. More importantly, we demonstrated that by upregulating PRDM1 expression, VLVs triggered differentiation signaling in targeted cells and facilitated viral lytic infection via activation of the RTA promoter. Our study not only demonstrates a new strategy for isolating VLVs but also shows the important roles of KSHV-associated VLVs in intercellular communication and the viral life cycle.

scholarly journals Modulation of oncogenic signaling networks by Kaposi’s sarcoma-associated herpesvirus

2017 ◽  
Vol 398 (8) ◽  
pp. 911-918 ◽  
Author(s):  
Jason P. Wong ◽  
Blossom Damania
Keyword(s):  
Life Cycle ◽  
Kaposi's Sarcoma ◽  
Primary Effusion Lymphoma ◽  
Kaposi’S Sarcoma ◽  
Host Cells ◽  
Signaling Networks ◽  
Oncogenic Signaling ◽  
Extracellular Signal ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Abstract Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of three human malignancies: Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. To persist and replicate within host cells, KSHV encodes proteins that modulate different signaling pathways. Manipulation of cell survival and proliferative networks by KSHV can promote the development of KSHV-associated malignancies. In this review, we discuss recent updates on KSHV pathogenesis and the viral life cycle. We focus on proteins encoded by KSHV that modulate the phosphatidylinositol-4,5-bisphosphate 3 kinase and extracellular signal-regulated kinases 1/2 pathways to create an environment favorable for viral replication and the development of KSHV malignancies.

scholarly journals K-bZIP of Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 (KSHV/HHV-8) Binds KSHV/HHV-8 Rta and Represses Rta-Mediated Transactivation

2003 ◽  
Vol 77 (6) ◽  
pp. 3809-3815 ◽  
Author(s):  
Wei Liao ◽  
Yong Tang ◽  
Su-Fan Lin ◽  
Hsing-Jien Kung ◽  
Chou-Zen Giam
Keyword(s):  
Viral Replication ◽  
Kaposi's Sarcoma ◽  
Leucine Zipper ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Early Gene ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8 ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The regulatory circuit for Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) gene expression bears resemblance to that of Epstein-Barr virus (EBV), but with interesting differences. Based on protein sequence similarities and synteny to their EBV counterparts, two KSHV/HHV-8 viral regulatory factors, HHV-8 Rta and K-bZIP, encoded by open reading frame (ORF) 50 and ORF K8, respectively, have been identified. Rta is an immediate early transcriptional activator that activates lytic viral replication and mediates viral reactivation from latency, while ORF K8 is an early gene activated by Rta. Extensive splicing of ORF K8 mRNA leads to the production of K-bZIP, a protein of the basic domain-leucine zipper (bZIP) family. The role of K-bZIP in viral replication, however, remains unresolved. Here, we report that K-bZIP is a nuclear protein that binds Rta directly both in vivo and in vitro and represses Rta-mediated transactivation of the K-bZIP promoter. We further demonstrate that the leucine zipper domain of K-bZIP is required for Rta binding and a K-bZIP mutant lacking the leucine zipper does not repress Rta activity. Finally, the K-bZIP-mediated repression of Rta transactivation cannot be restored by overexpression of the transcriptional coactivator p300 or the p300-CBP-associated factor, P/CAF. Our results suggest that K-bZIP is involved in a feedback circuit to turn off its own expression and possibly the expression of other early genes activated by Rta.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Interacts with Bromodomain Protein Brd4 on Host Mitotic Chromosomes

2006 ◽  
Vol 80 (18) ◽  
pp. 8909-8919 ◽  
Author(s):  
Jianxin You ◽  
Viswanathan Srinivasan ◽  
Gerald V. Denis ◽  
William J. Harrington ◽  
Mary E. Ballestas ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Host Cells ◽  
Nuclear Antigen ◽  
Mitotic Chromosomes ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Chromosome Binding

ABSTRACT The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is required for viral episome maintenance in host cells during latent infection. Two regions of the protein have been implicated in tethering LANA/viral episomes to the host mitotic chromosomes, and LANA chromosome-binding sites are subjects of high interest. Because previous studies had identified bromodomain protein Brd4 as the mitotic chromosome anchor for the bovine papillomavirus E2 protein, which tethers the viral episomes to host mitotic chromosomes (J. You, J. L. Croyle, A. Nishimura, K. Ozato, and P. M. Howley, Cell 117:349-360, 2004, and J. You, M. R. Schweiger, and P. M. Howley, J. Virol. 79:14956-14961, 2005), we examined whether KSHV LANA interacts with Brd4. We found that LANA binds Brd4 in vivo and in vitro and that the binding is mediated by a direct protein-protein interaction between the ET (extraterminal) domain of Brd4 and a carboxyl-terminal region of LANA previously implicated in chromosome binding. Brd4 associates with mitotic chromosomes throughout mitosis and demonstrates a strong colocalization with LANA and the KSHV episomes on host mitotic chromosomes. Although another bromodomain protein, RING3/Brd2, binds to LANA in a similar fashion in vitro, it is largely excluded from the mitotic chromosomes in KSHV-uninfected cells and is partially recruited to the chromosomes in KSHV-infected cells. These data identify Brd4 as an interacting protein for the carboxyl terminus of LANA on mitotic chromosomes and suggest distinct functional roles for the two bromodomain proteins RING3/Brd2 and Brd4 in LANA binding. Additionally, because Brd4 has recently been shown to have a role in transcription, we examined whether Brd4 can regulate the CDK2 promoter, which can be transactivated by LANA.

scholarly journals The Tudor SND1 protein is an m6A RNA reader essential for replication of Kaposi’s sarcoma-associated herpesvirus

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Belinda Baquero-Perez ◽  
Agne Antanaviciute ◽  
Ivaylo D Yonchev ◽  
Ian M Carr ◽  
Stuart A Wilson ◽  
...  
Keyword(s):  
Reading Ability ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Early Gene ◽  
Rna Modification ◽  
Royal Family ◽  
Cellular Mrnas ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

N6-methyladenosine (m6A) is the most abundant internal RNA modification of cellular mRNAs. m6A is recognised by YTH domain-containing proteins, which selectively bind to m6A-decorated RNAs regulating their turnover and translation. Using an m6A-modified hairpin present in the Kaposi’s sarcoma associated herpesvirus (KSHV) ORF50 RNA, we identified seven members from the ‘Royal family’ as putative m6A readers, including SND1. RIP-seq and eCLIP analysis characterised the SND1 binding profile transcriptome-wide, revealing SND1 as an m6A reader. We further demonstrate that the m6A modification of the ORF50 RNA is critical for SND1 binding, which in turn stabilises the ORF50 transcript. Importantly, SND1 depletion leads to inhibition of KSHV early gene expression showing that SND1 is essential for KSHV lytic replication. This work demonstrates that members of the ‘Royal family’ have m6A-reading ability, greatly increasing their epigenetic functions beyond protein methylation.

scholarly journals Activation of the Kaposi's Sarcoma-Associated Herpesvirus Major Latency Locus by the Lytic Switch Protein RTA (ORF50)

2005 ◽  
Vol 79 (13) ◽  
pp. 8493-8505 ◽  
Author(s):  
Satoko Matsumura ◽  
Yuriko Fujita ◽  
Evan Gomez ◽  
Naoko Tanese ◽  
Angus C. Wilson
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Early Gene ◽  
Viral Gene ◽  
Transcription Activator ◽  
Potent Inducer ◽  
Alternative Mrna Splicing ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) maintains a latent infection in primary effusion lymphoma cells but can be induced to enter full lytic replication by exposure to a variety of chemical inducing agents or by expression of the KSHV-encoded replication and transcription activator (RTA) protein. During latency, only a few viral genes are expressed, and these include the three genes of the so-called latency transcript (LT) cluster: v-FLIP (open reading frame 71 [ORF71]), v-cyclin (ORF72), and latency-associated nuclear antigen (ORF73). During latency, all three open reading frames are transcribed from a common promoter as part of a multicistronic mRNA. Subsequent alternative mRNA splicing and internal ribosome entry allows for the expression of each protein. Here, we show that transcription of LT cassette mRNA can be induced by RTA through the activation of a second promoter (LTi) immediately downstream of the constitutively active promoter (LTc). We identified a minimal cis-regulatory region, which overlaps with the promoter for the bicistronic K14/v-GPCR delayed early gene that is transcribed in the opposite direction. In addition to a TATA box at −30 relative to the LTi mRNA start sites, we identified three separate RTA response elements that are also utilized by the K14/v-GPCR promoter. Interestingly, LTi is unresponsive to sodium butyrate, a potent inducer of lytic replication. This suggests there is a previously unrecognized class of RTA-responsive promoters that respond to direct, but not indirect, induction of RTA. These studies highlight the fact that induction method can influence the precise program of viral gene expression during early events in reactivation and also suggest a mechanism by which RTA contributes to establishment of latency during de novo infections.

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