Article

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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    • "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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