ABSTRACT: 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.
