Genomewide Mapping and Screening of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) 3′ Untranslated Regions Identify Bicistronic and Polycistronic Viral Transcripts as Frequent Targets of KSHV MicroRNAs

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.

Section: Resultssupporting

confidence: 79%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Schifano

Corcoran

Kelkar

et al. 2017

“…Cells were centrifuged at 2,000 ϫ g for 1.0 h at room temperature with virus-containing supernatant and incubated at 37°C. The numbers of green fluorescent protein (GFP)-positive 293T cells representing infectious KSHV particles were easily visualized under a fluorescence microscope after 2 days postinfection, and virus-infected cells were quantitated by flow cytometry as previously described (30,31).…”

Section: Methodsmentioning

confidence: 99%

Identification of the Physiological Gene Targets of the Essential Lytic Replicative Kaposi's Sarcoma-Associated Herpesvirus ORF57 Protein

Verma

Krueger

et al. 2015

The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 gene product is essential for lytic KSHV replication and virion production. Recombinant ORF57-null mutants fail to accumulate several lytic cycle mRNAs at wild-type levels, leading to decreased production of lytic proteins necessary for efficient replication. Several mechanisms by which ORF57 may enhance expression of lytic KSHV mRNAs have been proposed, including mRNA stabilization, mRNA nuclear export, increased polyadenylation, and transcriptional activation. ORF57 activity is also gene specific, with some genes being highly dependent on ORF57, whereas others are relatively independent. Most experiments have utilized transfection models for ORF57 and have not systematically examined the gene specificity and potential mechanisms of action of ORF57 in the context of KSHV-infected cells. In this study, the KSHV genes that are most highly upregulated by ORF57 during KSHV lytic replication were identified by a combination of high-throughput deep RNA sequencing, quantitative PCR, Northern blotting, and rapid amplification of cDNA ends methods. Comparison of gene expression from a ⌬ORF57 KSHV recombinant, a rescued ⌬ORF57 KSHV recombinant, and wild-type KSHV revealed that two clusters of lytic genes are most highly dependent on ORF57 for efficient expression. Despite contiguous location in the genome and shared polyadenylation of several of the ORF57-dependent genes, ORF57 regulation was promoter and polyadenylation signal independent, suggesting that the mRNAs are stabilized by ORF57. The eight genes identified to critically require ORF57 belong to both early and late lytic temporal classes, and seven are involved in DNA replication, virion assembly, or viral infectivity, explaining the essential role of ORF57 in infectious KSHV production. IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus involved in the causation of several human cancers. The KSHV ORF57 protein is required for KSHV to replicate and produce infectious virus. We have identified several KSHV genes whose expression is highly dependent on ORF57 and shown that ORF57 increases expression of these genes specifically. These genes code for proteins that are required for the virus to replicate its DNA and to infect other cells. Identifying the targets and mechanism of action of ORF57 provides further approaches to discover antiviral therapy. K aposi's sarcoma associated herpesvirus (KSHV; human herpesvirus 8) is a human gammaherpesvirus associated with the development of Kaposi's sarcoma and several pathogenic lymphoproliferative syndromes (for a review, see reference 1). Lytic KSHV replication plays a role in pathogenesis by maintaining the infected cell reservoir, and lytic-phase KSHV proteins contribute to cell proliferation, inflammation, and immune evasion (2, 3). Understanding the mechanism of action of proteins required for lytic herpesvirus replication is therefore critical for further understanding of oncogenic mechanisms and the development of new antivi...

“…MicroRNAs have been implicated in differentiation, apoptosis, proliferation, and diseases such as cancer. In fact, both viral and cellular microRNAs have been shown to regulate KSHV latency (52,(61)(62)(63)(64)(65). Thus, we considered that microRNAs also mediate Nef suppression of KSHV lytic replication.…”

Section: Soluble Nef Protein Inhibits Kshv Lytic Replication Followinmentioning

confidence: 99%

Inhibition of Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication by HIV-1 Nef and Cellular MicroRNA hsa-miR-1258

Yan

Shen

et al. 2014

Self Cite

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several AIDS-related malignancies, including IMPORTANCEThis study found that Nef, a secreted HIV-1 protein, suppressed KSHV lytic replication to promote KSHV latency. Mechanistic studies indicated that a Nef-upregulated cellular miRNA, hsa-miR-1258, inhibits KSHV replication by directly targeting a seed sequence in the KSHV RTA 3=UTR. These results illustrate that, in addition to viral miRNAs, cellular miRNAs also play an important role in regulating the life cycle of KSHV. Overall, this is the first study to report the involvement of Nef in KSHV latency, implying its likely important role in the pathogenesis of AIDS-related malignancies.