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

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.
PLoS Pathogens (Impact Factor: 7.56). 03/2010; 6(3):e1000799. DOI: 10.1371/journal.ppat.1000799
Source: PubMed


The control of RNA stability is a key determinant in cellular gene expression. The stability of any transcript is modulated through the activity of cis- or trans-acting regulatory factors as well as cellular quality control systems that ensure the integrity of a transcript. As a result, invading viral pathogens must be able to subvert cellular RNA decay pathways capable of destroying viral transcripts. Here we report that the Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein binds to a unique KSHV polyadenylated nuclear RNA, called PAN RNA, and protects it from degradation by cellular factors. ORF57 increases PAN RNA levels and its effects are greatest on unstable alleles of PAN RNA. Kinetic analysis of transcription pulse assays shows that ORF57 protects PAN RNA from a rapid cellular RNA decay process, but ORF57 has little effect on transcription or PAN RNA localization based on chromatin immunoprecipitation and in situ hybridization experiments, respectively. Using a UV cross-linking technique, we further demonstrate that ORF57 binds PAN RNA directly in living cells and we show that binding correlates with function. In addition, we define an ORF57-responsive element (ORE) that is necessary for ORF57 binding to PAN RNA and sufficient to confer ORF57-response to a heterologous intronless beta-globin mRNA, but not its spliced counterparts. We conclude that ORF57 binds to viral transcripts in the nucleus and protects them from a cellular RNA decay pathway. We propose that KSHV ORF57 protein functions to enhance the nuclear stability of intronless viral transcripts by protecting them from a cellular RNA quality control pathway.

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    • "PAN RNA is an early gene product comprising nearly 80% of total polyadenylated cellular transcripts (~500,000 copies/cell). This high copy number is attributable to two features of the ~1.1-kb PAN transcript: MTA/ORF57 binding near the 5ʹ′ end and a triple-helical stabilization element (ENE) upstream of the 3ʹ′ polyA tail (Fig. 4) (Conrad and Steitz, 2005; Mitton-Fry et al., 2010; Sahin et al., 2010). Bioinformatic studies have revealed ENEs in genomic regions syntenic to the PAN locus in three other γ-herpesviruses—rhesus rhadinovirus (RRV), equine herpesvirus 2 (EHV2) and retroperitoneal fibromatosis-associated herpesvirus Macaca nemestrina (RFHVMn)—suggesting the presence of PAN RNA homologs [(Tycowski et al., 2012); unpublished data]. "
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    Genes & development 03/2015; 29(6):567-584. DOI:10.1101/gad.259077.115 · 10.80 Impact Factor
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    • "Mta enhances the accumulation of both viral (Kirshner et al., 2000; Han and Swaminathan, 2006; Majerciak et al., 2006a,c, 2008) and cellular (Gupta et al., 2000) RNAs in a gene-specific manner. Mta binds directly to RNAs in a sequence specific fashion to stabilize them or to promote escape from RNA silencing (Majerciak et al., 2006b; Nekorchuk et al., 2007; Sahin et al., 2010; Kang et al., 2011b; Massimelli et al., 2011; Sei and Conrad, 2011), enhances RNA splicing (Majerciak et al., 2008), shuttles between nuclei in heterokaryon assays (Bello et al., 1999), and promotes the nuclear export of un-spliced RNAs (Malik et al., 2004b; Boyne et al., 2008; Jackson et al., 2011; Majerciak et al., 2011; Taylor et al., 2011). Mta also stimulates mRNA translation (Nishimura et al., 2004; Boyne et al., 2010a). "
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    ABSTRACT: Viruses are obligate intracellular pathogens whose biological success depends upon replication and packaging of viral genomes, and transmission of progeny viruses to new hosts. The biological success of herpesviruses is enhanced by their ability to reproduce their genomes without producing progeny viruses or killing the host cells, a process called latency. Latency permits a herpesvirus to remain undetected in its animal host for decades while maintaining the potential to reactivate, or switch, to a productive life cycle when host conditions are conducive to generating viral progeny. Direct interactions between many host and viral molecules are implicated in controlling herpesviral reactivation, suggesting complex biological networks that control the decision. One viral protein that is necessary and sufficient to switch latent KSHV into the lytic infection cycle is called K-Rta. Rta is a transcriptional activator that specifies promoters by binding direct DNA directly and interacting with cellular proteins. Among these cellular proteins, binding of K-Rta to RBP-Jk is essential for viral reactivation.. In contrast to the canonical model for Notch signaling, RBP-Jk is not uniformly and constitutively bound to the latent KSHV genome, but rather is recruited to DNA by interactions with K-Rta. Stimulation of RBP-Jk DNA binding requires high affinity binding of Rta to repetitive and palindromic “CANT DNA repeats” in promoters, and formation of ternary complexes with RBP-Jk. However, while K-Rta expression is necessary for initiating KSHV reactivation, K-Rta’s role as the switch is inefficient. Many factors modulate K-Rta’s function, suggesting that KSHV reactivation can be significantly regulated post-Rta expression and challenging the notion that herpesviral reactivation is bistable. This review analyzes rapidly evolving research on KSHV K-Rta to consider the role of K-Rta promoter specification in regulating the progression of KSHV reactivation.
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    • "Previous studies showed that PAN RNA stability in the absence of ORF57 seems to be dependent on the activity of a cis-acting 79-nucleotide (nt) RNA element located in the 3' PAN, called ENE (expression and nuclear retention element)34-37. In the absence of the ENE, PAN's stability appears to rely on ORF57 binding to a 5' motif 38. In this report, we identified a 9-nt core motif in the 5' PAN region that is responsible for ORF57-mediated PAN accumulation. "
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    ABSTRACT: Kaposi sarcoma-associated herpesvirus (KSHV) ORF57, also known as Mta (mRNA transcript accumulation), enhances viral intron-less transcript accumulation and promotes splicing of intron-containing viral RNA transcripts. In this study, we identified KSHV PAN, a long non-coding polyadenylated nuclear RNA as a main target of ORF57 by a genome-wide CLIP (cross-linking and immunoprecipitation) approach. KSHV genome lacking ORF57 expresses only a minimal amount of PAN. In cotransfection experiments, ORF57 alone increased PAN expression by 20-30-fold when compared to vector control. This accumulation function of ORF57 was dependent on a structured RNA element in the 5' PAN, named MRE (Mta responsive element), but not much so on an ENE (expression and nuclear retention element) in the 3' PAN previously reported by other studies. We showed that the major function of the 5' PAN MRE is increasing the RNA half-life of PAN in the presence of ORF57. Further mutational analyses revealed a core motif consisting of 9 nucleotides in the MRE-II , which is responsible for ORF57 interaction and function. The 9-nt core in the MRE-II also binds cellular PABPC1, but not the E1B-AP5 which binds another region of the MRE-II. In addition, we found that PAN RNA is partially exportable in the presence of ORF57. Together, our data provide compelling evidence as to how ORF57 functions to accumulate a non-coding viral RNA in the course of virus lytic infection.
    International journal of biological sciences 10/2011; 7(8):1145-60. · 4.51 Impact Factor
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