Kaposi's Sarcoma-Associated Herpesvirus ORF57 Protein Binds and Protects a Nuclear Noncoding RNA from Cellular RNA Decay Pathways

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). 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.

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Kaposi’s Sarcoma-Associated Herpesvirus Fine-Tunes the Temporal Expression of Late Genes by Manipulating a Host RNA Quality Control Pathway

Journal of Virology ◽

10.1128/jvi.00287-20 ◽

2020 ◽

Vol 94 (14) ◽

Cited By ~ 2

Author(s):

Julio C. Ruiz ◽

Anne M. Devlin ◽

Jiwoong Kim ◽

Nicholas K. Conrad

Keyword(s):

Kaposi's Sarcoma ◽

Proper Time ◽

Kaposi’S Sarcoma ◽

Rna Decay ◽

Temporal Expression ◽

Viral Transcript ◽

Nuclear Rna ◽

Cryptic Transcription ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human oncogenic nuclear DNA virus that expresses its genes using the host cell transcription and RNA processing machinery. As a result, KSHV transcripts are subject to degradation by at least two host-mediated nuclear RNA decay pathways, the PABPN1- and poly(A) polymerase α/γ (PAPα/γ)-mediated RNA decay (PPD) pathway and an ARS2-dependent decay pathway. Here, we present global analyses of viral transcript levels to further understand the roles of these decay pathways in KSHV gene expression. Consistent with our recent report that the KSHV ORF57 protein increases viral transcript stability by impeding ARS2-dependent decay, ARS2 knockdown has only modest effects on viral gene expression 24 h after lytic reactivation of wild-type virus. In contrast, inactivation of PPD has more widespread effects, including premature accumulation of late transcripts. The upregulation of late transcripts does not require the primary late-gene-specific viral transactivation factor, suggesting that cryptic transcription produces the transcripts that then succumb to PPD. Remarkably, PPD inactivation has no effect on late transcripts at their proper time of expression. We show that this time-dependent PPD evasion by late transcripts requires the host factor nuclear RNAi-defective 2 (NRDE2), which has previously been reported to protect cellular RNAs by sequestering decay factors. From these studies, we conclude that KSHV uses PPD to fine-tune the temporal expression of its genes by preventing their premature accumulation. IMPORTANCE Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that causes Kaposi’s sarcoma and other lymphoproliferative disorders. Nuclear expression of KSHV genes results in exposure to at least two host-mediated nuclear RNA decay pathways, the PABPN1- and PAPα/γ-mediated RNA decay (PPD) pathway and an ARS2-mediated decay pathway. Perhaps unsurprisingly, we previously found that KSHV uses specific mechanisms to protect its transcripts from ARS2-mediated decay. In contrast, here we show that PPD is required to dampen the expression of viral late transcripts that are prematurely transcribed, presumably due to cryptic transcription early in infection. At the proper time for their expression, KSHV late transcripts evade PPD through the activity of the host factor NRDE2. We conclude that KSHV fine-tunes the temporal expression of its genes by modulating PPD activity. Thus, the virus both protects from and exploits the host nuclear RNA decay machinery for proper expression of its genes.

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PAN’s Labyrinth: Molecular Biology of Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) PAN RNA, a Multifunctional Long Noncoding RNA

Viruses ◽

10.3390/v6114212 ◽

2014 ◽

Vol 6 (11) ◽

pp. 4212-4226 ◽

Cited By ~ 33

Author(s):

Cyprian Rossetto ◽

Gregory Pari

Keyword(s):

Molecular Biology ◽

Long Noncoding Rna ◽

Noncoding Rna ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

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Kaposi’s sarcoma-associated herpesvirus fine-tunes the temporal expression of late genes by manipulating a host RNA quality control pathway

10.1101/2020.02.18.955526 ◽

2020 ◽

Author(s):

Julio C. Ruiz ◽

Anne Devlin ◽

Jiwoong Kim ◽

Nicholas K. Conrad

Keyword(s):

Kaposi's Sarcoma ◽

Proper Time ◽

Kaposi’S Sarcoma ◽

Rna Decay ◽

Temporal Expression ◽

Viral Transcript ◽

Nuclear Rna ◽

Cryptic Transcription ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

AbstractKaposi’s sarcoma-associated herpesvirus (KSHV) is a human oncogenic nuclear DNA virus that expresses its genes using the host cell transcription and RNA processing machinery. As a result, KSHV transcripts are subject to degradation by at least two host-mediated nuclear RNA decay pathways, PABPN1 and PAPα/γ-mediated RNA decay (PPD) and an ARS2-dependent decay pathway. Here, we present global analyses of viral transcript levels to further understand the roles of these decay pathways in KSHV gene expression. Consistent with our recent report that the KSHV ORF57 protein increases viral transcript stability by impeding ARS2-dependent decay, ARS2 knockdown has little effect on viral gene expression 24 hours after lytic reactivation of wild-type virus. In contrast, inactivation of PPD results in premature accumulation of late transcripts. The up-regulation of late transcripts does not require the primary late gene-specific viral transactivation factor, suggesting that cryptic transcription produces the transcripts that then succumb to PPD. Remarkably, PPD inactivation has no effect on late transcripts at their proper time of expression. We show that this time-dependent PPD evasion by late transcripts requires the host factor NRDE2, which has previously been reported to protect cellular RNAs by sequestering decay factors. From these studies, we conclude that KSHV uses PPD to fine-tune the temporal expression of its genes by preventing their premature accumulation.ImportanceKaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that causes Kaposi’s sarcoma and other lymphoproliferative disorders. Nuclear expression of KSHV genes results in exposure to at least two host-mediated nuclear RNA decay pathways, PABPN1 and PAPα/γ-mediated RNA decay (PPD) and an ARS2-mediated decay pathway. Perhaps unsurprisingly, we previously found that KSHV uses specific mechanisms to protect its transcripts from ARS2-mediated decay. In contrast, here we show that PPD is required to dampen the expression of viral late transcripts that are prematurely transcribed, presumably due to cryptic transcription early in infection. At the proper time for their expression, KSHV late transcripts evade PPD through the activity of the host factor NRDE2. We conclude that KSHV fine-tunes the temporal expression of its genes by modulating PPD activity. Thus, the virus both protects from and exploits the host nuclear RNA decay machinery for proper expression of its genes.

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