Expression of the Antisense-to-Latency Transcript Long Noncoding RNA in Kaposi's Sarcoma-Associated Herpesvirus

Abstract

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). Nextgeneration 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 ϳ10,000-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.

Kaposi’s Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions

“…PAN mainly localizes to the nucleus but can also be found in the cytoplasm and packaged into KSHV virions, where it may be active in newly infected cells as an immediate early RNA (57,60). The K7.3 transcript arising from the strand opposite PAN (25,30), has unknown function(s). Like PAN, K7.3 RNA is induced during virus replication, albeit at much lower abundance, and while it is assumed to be noncoding, this has not been formally examined (25).…”

“…Most KSHV lncRNAs other than PAN run antisense to known open reading frames (ORFs). Notable among these are the antisense-to-latency transcript (ALT), which is transcribed antisense to the major viral latency locus; T3.0 and T1.2, which are oriented opposite to replication and transcription activator (RTA/ORF50); and K7.3, which runs antisense to PAN (12,22,23,27,30).…”

Viral effects on the content and function of extracellular vesicles

“…More likely, these distinct effects identified in cultured cell lines represent different steps in natural infection perhaps by altering initial infection of different cell types, enhancing or limiting systemic spread throughout the host, or by altering viral virulence to facilitate persistence or establishment of latency. In sum, herpesviruses, by virtue of their large genomes (encoding > 100 proteins, as well as miRNAs and lncRNAs 154 ) modulate EV biogenesis and EV function through multiple independent mechanisms.…”

The Structure-to-Function Relationships of Gammaherpesvirus-Encoded Long Non-Coding RNAs and their Contributions to Viral Pathogenesis

“…Kaposi's sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma, as well as certain B-cell lymphomas, including primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD) [47]- [49]. KSHV encodes several lncRNAs [9], [50]- [53] ( Table 1), and PAN RNA is the most abundantly produced transcript during lytic reactivation [54]. PAN RNA has been shown to fold into three branched domains, each of which contains well-defined motifs connected by less structurally constrained regions (Figure 1) [55].…”

“…Although these three lncRNAs do not have canonical ORFs, all have been reported to be ribosome-associated, similar to PAN RNA [9]. ALT is a 10 Kb polyadenylated early lytic transcript expressed antisense to the major viral latency transcripts encoding LANA and the viral microRNAs [50]. In addition, ALT is on the same strand and is co-terminal with a bicistronic lytic transcript containing ORF K14 (v-OX2, a homolog of cellular surface receptor OX2) and ORF74 (vGPCR, viral G protein-coupled receptor) [67].…”