Charting Latency Transcripts in Kaposi's Sarcoma-Associated Herpesvirus by Whole-Genome Real-Time Quantitative PCR

Department of Microbiology and Immunology, The University of Oklahoma Health Science Center, Oklahoma City, Oklahoma 73104, USA.
Journal of Virology (Impact Factor: 4.65). 07/2002; 76(12):6213-23. DOI: 10.1128/JVI.76.12.6213-6223.2002
Source: PubMed

ABSTRACT The division into a latent or lytic life cycle is fundamental to all herpesviridae. In the case of Kaposi's sarcoma-associated herpesvirus (KSHV) (human herpesvirus 8), latent genes have been implicated in cell autonomous transformation, while certain lytic genes procure a tumor friendly milieu through paracrine mechanism. To query KSHV transcription, we devised and validated a high-throughput, high-specificity, high-sensitivity, real-time quantitative reverse transcription-PCR array. This novel methodology is applicable to many human pathogens. Its first use demonstrated that the mRNA levels for KSHV LANA, v-cyclin, and v-FLIP do not increase at any time after viral reactivation. The mRNA for LANA-2/vIRF-3 is similarly resistant to viral reactivation. In contrast, every other latent or lytic message was induced. Hence, LANA, v-FLIP, v-cyclin, and LANA-2 constitute a group of uniquely regulated transcripts in the KSHV genome.

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Available from: Dirk P Dittmer, Aug 09, 2015
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    • "This platform is flexible such that individual QPCR assays can easily be substituted according to the needs of a particular experiment and expanded to a 96 Â 96 format if screening for additional transcripts is required in the future. While PCR-based array profiling of KSHV and EBV transcripts in primary Kaposi sarcoma lesions and virus-positive cell lines has been reported previously (Dittmer, 2003; Fakhari and Dittmer, 2002; Kurokawa et al., 2005; Wang et al., 2009; Whitehurst et al., 2013), the present work provides two novel features. First, the Fluidigm system requires very little starting material (less than 25 ng RNA) making it suitable for analyzing small clinical samples. "
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    ABSTRACT: We have validated a flexible, high-throughput and relatively inexpensive RT-QPCR array platform for absolute quantification of Epstein–Barr virus transcripts in different latent and lytic infection states. Several novel observations are reported. First, during infection of normal B cells, Wp-initiated latent gene transcripts remain far more abundant following activation of the Cp promoter than was hitherto suspected. Second, EBNA1 transcript levels are remarkably low in all forms of latency, typically ranging from 1 to 10 transcripts per cell. EBNA3A, -3B and -3C transcripts are likewise very low in Latency III, typically at levels similar to or less than EBNA1 transcripts. Thirdly, a subset of lytic gene transcripts is detectable in Burkitt lymphoma lines at low levels, including: BILF1, which has oncogenic properties, and the poorly characterized LF1, LF2 and LF3 genes. Analysis of seven African BL biopsies confirmed this transcription profile but additionally revealed significant expression of LMP2 transcripts.
    Virology 11/2014; 474. DOI:10.1016/j.virol.2014.10.030 · 3.28 Impact Factor
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    • "The K13 (ORF71) latent viral gene encodes viral FLICE (Fas-associated death-domain like IL-1 β-converting enzyme) inhibitory protein (vFLIP), a truncated homolog of cellular FLIP (cFLIP), (Dittmer et al., 1998; Fakhari and Dittmer, 2002). The vFLIP gene is expressed in KS and PEL cells from a polycistronic mRNA encompassing the latency locus. "
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    ABSTRACT: Kaposi's sarcoma-associated herpesvirus (KSHV), a member of the herpesvirus family, has evolved to establish a long-term, latent infection of cells such that while they carry the viral genome gene expression is highly restricted. Latency is a state of cryptic viral infection associated with genomic persistence in their host and this hallmark of KSHV infection leads to several clinical-epidemiological diseases such as KS, a plasmablastic variant of multicentric Castleman's disease, and primary effusion lymphoma upon immune suppression of infected hosts. In order to sustain efficient life-long persistency as well as their life cycle, KSHV dedicates a large portion of its genome to encode immunomodulatory proteins that antagonize its host's immune system. In this review, we will describe our current knowledge of the immune evasion strategies employed by KSHV at distinct stages of its viral life cycle to control the host's immune system.
    Frontiers in Microbiology 03/2012; 3:44. DOI:10.3389/fmicb.2012.00044 · 3.94 Impact Factor
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    • "In a cell latently infected with Kaposi's sarcoma-associated herpesvirus (KSHV), the switch to viral lytic replication can be achieved via signals transmitted from interferon-γ, phorbol ester, HDAC inhibitors, or with the ectopic expression of KSHV replication and transcription activator, referred to as K-RTA (also known as ORF50 and Lyta, reviewed in Ganem, 2007). Consecutive expression of KSHV lytic genes induced by either chemicals or K-RTA has been previously demonstrated (Sarid et al., 1998; Lukac et al., 1999; Sun et al., 1999; Gradoville et al., 2000; Fakhari and Dittmer, 2002; Nakamura et al., 2003; Yoo et al., 2005). In addition, the combination of sodium butyrate plus ectopic K-RTA yielded synergistic effects in an HEK293 cell background (Vieira and O'Hearn, 2004). "
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    ABSTRACT: The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV), K-RTA, is a lytic switch protein that moderates the reactivation process of KSHV latency. By mass spectrometric analysis of affinity purified K-RTA, we showed that Thr-513 or Thr-514 was the primary in vivo phosphorylation site. Thr-513 and Thr-514 are proximal to the nuclear localization signal ((527)KKRK(530)) and were previously hypothesized to be target sites of Ser/Thr kinase hKFC. However, substitutions of Thr with Ala at 513 and 514 had no effect on K-RTA subcellular localization or transactivation activity. By contrast, replacement of Ser with Ala at Ser-634 and Ser-636 located in a Ser/Pro-rich region of K-RTA, designated as S634A/S636A, produced a polypeptide with ∼10 kDa shorter in molecular weight and reduced transactivation in a luciferase reporter assay relative to the wild type. In contrast to prediction, the decrease in molecular weight was not due to lack of phosphorylation because the overall Ser and Thr phosphorylation state in K-RTA and S634A/S636A were similar, excluding that Ser-634 or Ser-636 motif served as docking sites for consecutive phosphorylation. Interestingly, S634A/S636A lost ∼30% immuno-reactivity to MPM2, an antibody specific to pSer/pThr-Pro motif, indicating that (634)SPSP(637) motif was in vivo phosphorylated. By in vitro kinase assay, we showed that K-RTA is a substrate of CDK9, a Pro-directed Ser/Thr kinase central to transcriptional regulation. Importantly, the capability of K-RTA in associating with endogenous CDK9 was reduced in S634A/S636A, which suggested that Ser-634 and Ser-636 may be involved in CDK9 recruitment. In agreement, S634A/S636A mutant exhibited ∼25% reduction in KSHV lytic cycle reactivation relative to that by the wild type K-RTA. Taken together, our data propose that Ser-634 and Ser-636 of K-RTA are phosphorylated by host transcriptional kinase CDK9 and such a process contributes to a full transcriptional potency of K-RTA.
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