The regulation of latency is central to herpesvirus biology. Recent transcriptome-wide surveys have uncovered evidence for promiscuous transcription across the entirety of the Kaposi sarcoma-associated herpesvirus genome and postulated the existence of multiple viral long noncoding RNAs (lncRNAs). NextGen sequencing studies are highly dependent on the specific experimental approach and particular algorithms of analysis, and therefore benefit from independent confirmation of the results. The antisense to latency transcripts (ALT) lncRNA was discovered by genome-tiling microarray (Chandriani et al. J. Virol. 16: 7934-7942, 2010, doi: 10.1128/JVI.00645-10). To characterize ALT in detail, we physically isolated this lncRNA by a strand-specific hybrid-capture assay and then employed RNA-seq and novel RT-PCR assays to distinguish all RNA species in the KSHV latency region. These methods confirm that ALT initiates at 120,739/121,012 and encodes a single splice site, which is shared with the 3′ -coterminal K14-vGPCR/ORF74, terminating at 130,873 (GQ994935), resulting in a ∼10,000-nt transcript. No shorter ALT isoforms were identified. This study also identified a novel intron within the LANA 5′ UTR using a splice acceptor at 127,888. In sum, ALT joins PAN/nut1/T1.1 as a bona fide lncRNA of KSHV with potentially important roles in viral gene regulation and pathogenesis.
Increasing data support the importance of noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), which have been shown to exert critical regulatory functions without coding for recognizable proteins. Defining the sequences of these ncRNAs is essential for future studies aiming to functionally characterize a specific ncRNA. Most lncRNA studies are highly dependent on high-throughput sequencing and bioinformatic analyses, few studies follow up on the initial predictions, and analyses are at times discordant. This manuscript characterizes one key viral lncRNA, ALT, by physically isolating ALT and by a sequencing-independent assay. It provides for a simple assay to follow lncRNA expression in experimental and clinical samples. ALT is expressed antisense to the major viral latency transcripts encoding LANA as well as the viral microRNAs, and thus has the potential to regulate this key part of the viral life cycle.