scholarly journals Transcription Mapping and Expression Patterns of Genes in the Major Immediate-Early Region of Kaposi's Sarcoma-Associated Herpesvirus

Virology ◽  
2002 ◽  
Vol 299 (2) ◽  
pp. 301-314 ◽  
Author(s):  
Alexei K. Saveliev ◽  
Fan Xiu Zhu ◽  
Yan Yuan
Keyword(s):  
Kaposi's Sarcoma ◽  
Expression Patterns ◽  
Kaposi’S Sarcoma ◽  
Immediate Early ◽  
Early Region ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

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 Asynchronous Progression through the Lytic Cascade and Variations in Intracellular Viral Loads Revealed by High-Throughput Single-Cell Analysis of Kaposi's Sarcoma-Associated Herpesvirus Infection

2006 ◽  
Vol 80 (20) ◽  
pp. 10073-10082 ◽  
Author(s):  
Laura A. Adang ◽  
Christopher H. Parsons ◽  
Dean H. Kedes
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
High Throughput ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Immediate Early ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus-8) is frequently tumorigenic in immunocompromised patients. The average intracellular viral copy number within infected cells, however, varies markedly by tumor type. Since the KSHV-encoded latency-associated nuclear antigen (LANA) tethers viral episomes to host heterochromatin and displays a punctate pattern by fluorescence microscopy, we investigated whether accurate quantification of individual LANA dots is predictive of intracellular viral genome load. Using a novel technology that integrates single-cell imaging with flow cytometry, we found that both the number and the summed immunofluorescence of individual LANA dots are directly proportional to the amount of intracellular viral DNA. Moreover, combining viral (immediate early lytic replication and transcription activator [RTA] and late lytic K8.1) and cellular (syndecan-1) staining with image-based flow cytometry, we were also able to rapidly and simultaneously distinguish among cells supporting latent, immediate early lytic, early lytic, late lytic, and a potential fourth “delayed late” category of lytic replication. Applying image-based flow cytometry to KSHV culture models, we found that de novo infection results in highly varied levels of intracellular viral load and that lytic induction of latently infected cells likewise leads to a heterogeneous population at various stages of reactivation. These findings additionally underscore the potential advantages of studying KSHV biology with high-throughput analysis of individual cells.

scholarly journals Identification of the Immediate-Early Transcripts of Kaposi’s Sarcoma-Associated Herpesvirus

1999 ◽  
Vol 73 (7) ◽  
pp. 5556-5567 ◽  
Author(s):  
Fan Xiu Zhu ◽  
Teresa Cusano ◽  
Yan Yuan
Keyword(s):  
Protein Synthesis ◽  
Kaposi's Sarcoma ◽  
Screening Method ◽  
Kaposi’S Sarcoma ◽  
Viral Reactivation ◽  
Immediate Early ◽  
Transcription Activator ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Inhibitor Of Protein Synthesis ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT In the immediate-early phase of reactivation or primary infection, herpesviruses express a small number of genes without requiring prior viral protein synthesis. Immediate-early genes usually encode regulatory proteins critical for the viral life cycle. Kaposi’s sarcoma-associated herpesvirus (KSHV) gene transcription in the immediate-early stage of viral reactivation was examined by using a chemical induction combined with a gene expression screening method. RNA transcripts from at least four KSHV genomic loci accumulate when latently infected B-lymphoma cells are induced for reactivation in the presence of an inhibitor of protein synthesis (cycloheximide) and thus represent immediate-early class transcripts. Among them, a 3.6-kb mRNA encodes three putative open reading frames (ORFs), namely, ORF50, K8, and K8.2. ORF50 is a homologue of Rta, a transcription activator encoded by Epstein-Barr virus (EBV). The K8 gene codes for a 237-amino-acid protein with a basic-leucine zipper domain near its C terminus and an acidic domain near its N terminus and which closely resembles the ZEBRA protein of EBV and Jun/Fos family proteins. Other immediate-early mRNAs of KSHV include a 1.7-kb mRNA encoding ORF45, a 2.0-kb mRNA encoding ORF K4.2, and a 4.5-kb mRNA. Functional roles of products of these KSHV immediate-early transcripts remain to be studied.

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 Kaposi's Sarcoma-Associated Herpesvirus Virion-Induced Transcription Activation of the ORF50 Immediate-Early Promoter

2005 ◽  
Vol 79 (20) ◽  
pp. 13180-13185 ◽  
Author(s):  
Fang Lu ◽  
Latasha Day ◽  
Paul M. Lieberman
Keyword(s):  
Kaposi's Sarcoma ◽  
Core Promoter ◽  
Protein Composition ◽  
Kaposi’S Sarcoma ◽  
Transcription Activation ◽  
Lytic Cycle ◽  
Immediate Early ◽  
Mobility Shift ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Lytic cycle reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) can be initiated by transcription activation of the ORF50 immediate-early (IE) gene promoter (ORF50p). We provide evidence that KSHV virions stimulate transcription of ORF50p. Virion activation was resistant to UV inactivation and cycloheximide treatment. The virion-responsive element was mapped to core promoter region −150 to + 1 relative to the ORF50 initiation codon. Electrophoretic mobility shift assays and chromatin immunoprecipitation suggest that KSHV virions indirectly alter the protein composition and chromatin modifications at ORF50p. These data suggest that KSHV virions possess an IE trans-inducing function similar to that observed in alpha- and betaherpesviruses.

scholarly journals A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

2018 ◽  
Vol 92 (17) ◽  
Author(s):  
Allison Alwan TerBush ◽  
Florianne Hafkamp ◽  
Hee Jun Lee ◽  
Laurent Coscoy
Keyword(s):  
Kaposi's Sarcoma ◽  
Expression Patterns ◽  
Kaposi’S Sarcoma ◽  
Cell Types ◽  
Eph Receptors ◽  
Kshv Infection ◽  
Receptor Usage ◽  
Infection Mechanism ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTHost receptor usage by Kaposi's sarcoma-associated herpesvirus (KSHV) has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infections. In these two infection models, KSHV adheres to the cell though heparan sulfate (HS) binding and then interacts with a complex of EphA2, xCT, and integrins α3β1, αVβ3, and αVβ5 to catalyze viral entry. We dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS and EphA2 but is independent of αV- and β1-family integrin expression. Furthermore, infection appears to be independent of the EphA2 intracellular domain. We also demonstrated that while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2.IMPORTANCEOur data reveal an integrin-independent route of KSHV infection and suggest that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph receptors are large protein families with diverse expression patterns across cells and tissues, we propose that KSHV may engage with several proteins from both families in different combinations to negotiate successful entry into diverse cell types.

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