Project description:Purpose: To characterize genome-wide microRNA (miRNA) expression profiles in the context of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic reactivation. Methods: We utilized a well-established model of KSHV reactivation, the doxycycline (Dox)-inducible KSHV producer cell line iSLK.219, which contains a latent KSHV genome and a Dox-inducible KSHV lytic switch protein RTA (replication and transcription activator) to mediate efficient reactivation upon Dox treatment. We performed small-RNA sequencing of RNA isolated from latent KSHV-infected iSLK.219 cells at 0 h or lytic iSLK.219 cells at 72 h post Dox-induced KSHV lytic reactivation using Illumina HiSeq 2500. To rule out the possibility that Dox itself can affect miRNA expression, we also performed small-RNA sequencing of RNA isolated from KSHV-negative iSLK cells, which lacks the KSHV genome but harbors the Dox-inducible RTA transgene, without Dox treatment at 0 h or at 72 h post Dox treatment. The mappable reads were aligned to the human genome and miRBase using Bowtie. Results: Global cellular miRNA transcriptome analysis has identified changes in the host miRNA expression landscape during the switch from latent to lytic KSHV replication. The top down-regulated miRNAs included miR-31-5p, miR-29a-3p, miR-181a-3p, miR-194-5p, and miR-449c-5p and the top up-regulated miRNAs included miR-139-5p, miR-7-5p, miR-210-3p and miR-3065-5p. We confirmed that Dox treatment in KSHV-negative iSLK cells did not significantly down-regulate or up-regulate these cellular miRNAs, indicating that Dox itself or RTA transgene expression alone does not affect the levels of these miRNAs. In addition, 8 KSHV-encoded miRNAs, including pre-miR-K12-12, were significantly up-regulated upon lytic reactivation in iSLK.219 cells. Notably, the top four up-regulated host miRNAs have been implicated in inflammatory signaling pathways involved in a productive or lytic cycle of infection, suggesting that KSHV exploits these immunomodulatory miRNAs to facilitate lytic reactivation. On the contrary, four out of the top five down-regulated host miRNAs, miR-29a-3p, miR-181a-3p, miR-194-5p, and 449c-5p have been implicated in restricting the infection and replication of RNA and DNA viruses. Taken together with our small-RNA sequencing analysis, these findings suggest that KSHV lytic reactivation impacts the cellular miRNA expression landscape as a strategy to evade or subvert host antiviral responses and ensure efficient lytic replication and persistence. Conclusions: Our study represents the first detailed analysis of genome-wide miRNA transcriptomes during KSHV lytic reactivation, with biological replicates, generated by miRNA-seq technology. Our study provides crucial insights into the impact of lytic KSHV infection on the host and viral miRNA expression landscapes. Moreover, our results identify a previously unappreciated role for miR-31-5p in regulating KSHV lytic reactivation by modulating KHDRBS3 expression.

Project description:The Kaposi's sarcoma associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi's sarcoma, primary effusion lymphoma (PEL), and some forms of multicentric Castleman's disease. The KSHV ORF57 protein is a conserved posttranscriptional regulator of gene expression that is essential for virus replication. ORF57 is multifunctional, but most of its activities are directly linked to its ability to bind RNA. We globally identified virus and host RNAs bound by ORF57 during lytic reactivation in PEL cells using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). As expected, ORF57-bound RNA fragments mapped throughout the KSHV genome, including the known ORF57 ligand PAN RNA. In agreement with previously published ChIP results, we observed that ORF57 bound RNAs near the oriLyt regions of the genome. Examination of the host RNA fragments revealed that a subset of the ORF57-bound RNAs was derived from transcript 5´ ends. The position of these 5´-bound fragments correlated closely with the 5´-most exonintron junction of the pre-mRNA. We selected four candidates (BTG1, EGR1, ZFP36, and TNFSF9) and analyzed their pre-mRNA and mRNA levels during lytic phase. Analysis of both steady-state and newly made RNAs revealed that these candidate ORF57-bound pre-mRNAs persisted for longer periods of time throughout infection than control RNAs, consistent with a role for ORF57 in pre-mRNA metabolism. In addition, exogenous expression of ORF57 was sufficient to increase the pre-mRNA levels and, in one case, the mRNA levels of the putative ORF57 targets. These results demonstrate that ORF57 interacts with specific host pre-mRNAs during lytic reactivation and alters their processing, likely by stabilizing pre-mRNAs. These data suggest that ORF57 is involved in modulating host gene expression in addition to KSHV gene expression during lytic reactivation. HITS-CLIP was performed on TREx BCBL-Rta cells 20 hpi using antibodies against ORF57. Three biological replicates were performed.

Project description:Aim: Circular RNA (circRNA) is a class of noncoding, single-stranded RNA generated by backsplicing, a process where the 5’ and 3’ ends of an RNA are covalently joined. Virally encoded circRNAs have been identified in several members of Gammaherpesvirinae, including Kaposi’s sarcoma-associated herpesvirus (KSHV). In KSHV, the viral interferon regulatory factor 4 (vIRF4) region produces two isoforms of circRNA (circ-vIRF4) that are detectable during latency and reactivation. Since these circRNAs are expressed during latency, a period where viral transcription is tightly restricted and tumorigenesis occurs, circ-vIRF4 may contribute to the development of KSHV malignancies. Therefore, the aim of this study is to characterize the function of vIRF4 circRNAs. Methods: A KSHV mutant (Δcirc-vIRF4) was generated in the BAC16 bacmid and transfected into 293T and iSLK cells. Expression of circRNA after mutagenesis was assessed by qualitative and quantitative PCR. Host and viral gene expression in iSLK cells during both viral latency and reactivation were also assessed by RNA-seq. Results: RT-PCR of Δcirc-vIRF4-infected iSLK cells demonstrated no expression of wild type isoforms, but PCR cloning showed that alternative backsplice sites were used to express novel vIRF4 circRNAs, where the most prominent isoform was a 1,020 nt isoform. RNA-seq analyses comparing WT- and Δcirc-vIRF4-infected iSLK cells demonstrated significant differential expression of both host and viral genes during both phases of the viral life cycle. Gene ontology analyses returned terms related to cell adhesion, proliferation, and migration for both datasets, as well as kinase signaling and apoptosis for the lytic dataset. Conclusions: These results show that KSHV can switch to an alternative backsplice site for vIRF4 circRNA production in the absence of a canonical splice site and that circ-vIRF4 contributes to the regulation of both host and viral gene expression through an unknown mechanism.

			Action	DRS
	SRR19793302.fastq.gz	Fastqsanger.gz
	SRR19793303.fastq.gz	Fastqsanger.gz

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