Kaposi's sarcoma-associated herpesvirus immediate early gene activity

10.2741/1394 ◽  
2004 ◽  
Vol 9 (1-3) ◽  
pp. 2245 ◽  
Author(s):  
Vincent Lacoste
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Immediate Early Gene ◽  
Gene Activity ◽  
Early Gene ◽  
Immediate Early ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

scholarly journals Identification and Analysis of the K5 Gene of Kaposi's Sarcoma-Associated Herpesvirus

2000 ◽  
Vol 74 (6) ◽  
pp. 2867-2875 ◽  
Author(s):  
Muzammel Haque ◽  
Jiguo Chen ◽  
Keiji Ueda ◽  
Yasuko Mori ◽  
Kazusi Nakano ◽  
...  
Keyword(s):  
Gene Product ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Northern Blotting ◽  
Early Gene ◽  
Immediate Early ◽  
Protein Synthesis Inhibitors ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8), belongs to the gammaherpesvirus subfamily and encodes ∼80 open reading frames (ORFs). Among them are a few candidates for immediate-early genes (e.g., K5). We developed a monoclonal antibody (MAb), 328C7, against the K5 antigen. This MAb reacted with the K5 gene product by immunoscreening of a cDNA library from BCBL-1 cells, and this result was confirmed by transfection of the K5 ORF into Cos-7 cells. After induction of lytic infection by treatment with 12-O-tetradecanoylphorbol-13-acetate, MAb 328C7 reacted with an antigen in the cytoplasm of BCBL-1 and BC-3 cells as early as after 4 h of induction. Immunoelectron microscopy showed that the K5 antigen was situated mainly in the endoplasmic reticulum but was not present on the virion or in the nucleus. Northern blotting with a K5-specific probe revealed a single transcript of 1.2 kb, while Western blotting showed the antigen to be a 36-kDa polypeptide. The 5′ and 3′ ends were then determined by rapid amplification of cDNA, followed by sequencing of RACE products, and a splice was revealed upstream of the K5 ORF. K5 expression was unaffected by the respective DNA and protein synthesis inhibitors phosphonoformic acid and cycloheximide plus actinomycin D, confirming its immediate-early nature. Transient-transfection assays showed that the K5 promoter was transactivated by ORF 50 (KSHV Rta), a homolog of Epstein-Barr virus Rta, but the K5 gene product exhibited no transregulation of its own promoter or those of DNA polymerase and the human immunodeficiency virus type 1 long terminal repeat. This is the first such analysis of an immediate-early gene product; determination of its specific biological function requires further investigation.

scholarly journals Auto-activation of the rta gene of human herpesvirus-8/Kaposi’s sarcoma-associated herpesvirus

2000 ◽  
Vol 81 (12) ◽  
pp. 3043-3048 ◽  
Author(s):  
Hongyu Deng ◽  
Arthur Young ◽  
Ren Sun
Keyword(s):  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Immediate Early Gene ◽  
Early Gene ◽  
Immediate Early ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8 ◽  
Latently Infected ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Rta, mainly encoded by open reading frame 50 (ORF50), is the product of an immediate-early gene of human herpesvirus-8 (HHV-8)/Kaposi’s sarcoma-associated herpesvirus. Rta is a transcriptional activator that is both necessary and sufficient to disrupt viral latency and activate the expression of downstream viral lytic genes. We report that ectopically expressed Rta protein could also activate the rta promoter on a reporter plasmid up to 144-fold, both in latently infected B cells and in uninfected epithelial cells, and that this activation was dose-dependent. Furthermore, by analysing the 5′ untranslated region using ribonuclease protection assays, we demonstrated that transfection of an Rta expression plasmid into latently infected cells activated the expression of rta transcripts from endogenous viral genomes. We propose that auto-activation of the immediate-early gene, rta, is an important strategy for HHV-8 to effectively respond to environmental stimuli and maximally activate the virus lytic cycle.

scholarly journals Kinetics of Kaposi’s Sarcoma-Associated Herpesvirus Gene Expression

1999 ◽  
Vol 73 (3) ◽  
pp. 2232-2242 ◽  
Author(s):  
Ren Sun ◽  
Su-Fang Lin ◽  
Katherine Staskus ◽  
Lyndle Gradoville ◽  
Elizabeth Grogan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Immediate Early Gene ◽  
Lytic Cycle ◽  
Early Gene ◽  
Immediate Early ◽  
Early Genes ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Herpesvirus gene expression can be classified into four distinct kinetic stages: latent, immediate early, early, and late. Here we characterize the kinetic class of a group of 16 Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 genes in a cultured primary effusion cell line and examine the expression of a subset of these genes in KS biopsies. Expression of two latent genes, LANA and vFLIP, was constitutive and was not induced by chemicals that induce the lytic cycle in primary effusion lymphoma (PEL) cell lines. An immediate-early gene, Rta (open reading frame 50 [ORF50]), was induced within 4 h of the addition of n-butyrate, and its 3.6-kb mRNA was resistant to inhibition by cycloheximide. Early genes, including K3 and K5 that are homologues of the “immediate-early” gene of bovine herpesvirus 4, K8 that is a positional homologue of Epstein-Barr virus BZLF1, vMIP II, vIL-6, and polyadenylated nuclear (PAN) RNA, appeared 8 to 13 h after chemical induction. A second group of early genes that were slightly delayed in their appearance included viral DHFR, thymidylate synthase, vMIP I, G protein-coupled receptor, K12, vBcl2, and a lytic transcript that overlapped LANA. The transcript of sVCA (ORF65), a late gene whose expression was abolished by Phosphonoacetic acid, an inhibitor of KSHV DNA replication, did not appear until 30 h after induction. Single-cell assays indicated that the induction of lytic cycle transcripts resulted from the recruitment of additional cells into the lytic cycle. In situ hybridization of KS biopsies showed that about 3% of spindle-shaped tumor cells expressed Rta, ORF K8, vIL-6, vMIP I, vBcl-2, PAN RNA, and sVCA. Our study shows that several KSHV-encoded homologues of cellular cytokines, chemokines, and antiapoptotic factors are expressed during the viral lytic cycle in PEL cell lines and in KS biopsies. The lytic cycle of KSHV, probably under the initial control of the KSHV/Rta gene, may directly contribute to tumor pathogenesis.

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.

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 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 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.

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