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's sarcoma-associated herpesvirus (KSHV) genome and postulated the existence of multiple viral long noncoding RNAs (lncRNAs). Next-generation 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 transcript (ALT) lncRNA was discovered by genome-tiling microarray (Chandriani et al., J Virol 86:7934-7942, 2010, https://doi.org/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 transcriptome sequencing and novel reverse transcription-PCR (RT-PCR) assays to distinguish all RNA species in the KSHV latency region. These methods confirm that ALT initiates at positions 120739/121012 and encodes a single splice site, which is shared with the 3'-coterminal K14-vGPCR/ORF74 mRNA, terminating at 130873 (GenBank accession number GQ994935), resulting in an ∼10,000-nucleotide transcript. No shorter ALT isoforms were identified. This study also identified a novel intron within the LANA 5' untranslated region using a splice acceptor at 127888. In summary, ALT joins PAN/nut1/T1.1 as a bona fide lncRNA of KSHV with potentially important roles in viral gene regulation and pathogenesis.

Importance: Increasing data support the importance of noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and 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. The manuscript characterizes one key viral lncRNA, ALT, by physically isolating ALT and by a sequencing-independent assay. It provides for a simple assay to monitor lncRNA expression in experimental and clinical samples. ALT is expressed antisense to the major viral latency transcripts encoding LANA as well as the viral miRNAs and thus has the potential to regulate this key part of the viral life cycle.