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

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.

Transcription mapping of human herpesvirus 8 genes encoding viral interferon regulatory factors

“…Lubyova & Pitha (2000) and Jenner et al (2001) characterized vIRF-3 as inducible. In contrast, Rivas et al (2001) and Fakhari & Dittmer (2002) showed that the gene is expressed constitutively. Lubyova & Pitha (2000) noted a potential TATA box and polyadenylation signal in the DNA sequence, but the 59-end of a single cDNA clone analysed by Rivas et al (2001) does not correspond with use of this TATA box.…”

“…Jenner et al (2001) detected the spliced transcript by RT-PCR, whereas Burysek & Pitha (2001) excluded splicing by similar experiments. Moreover, in contrast to Burysek et al (1999b) and Burysek & Pitha (2001), Jenner et al (2001) and Fakhari & Dittmer (2002) catalogued vIRF-2 as an inducible gene from microarray and quantitative PCR studies, respectively. In another microarray study, Paulose-Murphy et al (2001) noted that K11, which forms the second exon of vIRF-2, is inducible.…”

“…The splicing pattern of vIRF-4 has been demonstrated (Jenner et al, 2001), and the transcript characterized as inducible (Sarid et al, 1998;Paulose-Murphy et al, 2001;Jenner et al, 2001;Fakhari & Dittmer, 2002). The size of the mRNA and the locations of its ends have not been determined.…”

Regulation and Autoregulation of the Promoter for the Latency-associated Nuclear Antigen of Kaposi's Sarcoma-associated Herpesvirus

“…During lytic replication, all viral proteins are expressed and viral particles are released from the host cell, eventually resulting in the destruction of the host cell. During latent infection, only a few viral genes are expressed, and these are required for the establishment and maintenance of latency (13)(14)(15)(16)(17)(18). In KS, KSHV persists latently in the majority of tumor cells, with fewer than 5% of cells undergoing spontaneous lytic replication (19 -22).…”

Development of a fluorescence-based assay to screen antiviral drugs against Kaposi's sarcoma–associated herpesvirus

“…Real-time quantitative PCR (RT-QPCR) was done with primers specific for the cellular U6 gene and the viral vGPCR gene as previously described (33). Using this method to analyze BCBL-1 cells serially diluted in a suspension of uninfected BJAB cells, we were able to detect as few as 1 in 10,000 infected cells with 95% efficiency (Supplementary Fig.…”