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Genome-wide transcriptome analysis upon KSHV lytic reactivation

ABSTRACT: Purpose: To characterize genome-wide mRNA 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 and entry into the lytic cycle upon Dox treatment. We performed mRNA-sequencing (mRNA-seq) 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. The mappable reads were aligned to the human genome or the KSHV genome using Bowtie. Results: We analyzed global transcriptome changes in iSLK.219 cells during the latency to lytic transition and found that reactivated lytic iSLK.219 cells exhibited an increased abundance of cellular genes involved in cell cycle, DNA replication, and nuclear division, consistent with lytic infection activating mitogenic signaling to support viral DNA replication. In addition, expression of key viral lytic genes with strong mitogenic activities, including vIL-6 (K2), K1, and the KSHV-encoded chemokines, vCCL1 (K6), vCCL2 (K4) and vCCL3 (K4.1), were markedly induced during lytic KSHV replication. Conclusions: These results confirm that the gene expression profile of reactivated lytic iSLK.219 cells exhibit some key hallmark features of KSHV lytic reactivation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE281293 | GEO | 2026/02/24

REPOSITORIES: GEO

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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.

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Genome-wide transcriptome analysis upon KSHV lytic reactivation

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