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N6-methyladenosine modification in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection [MeRIP-seq]

ABSTRACT: Methylation at the N6 position of adenosine (m6A) is a highly prevalent reversible modification within eukaryotic mRNAs that has been linked to many stages of RNA processing and fate. Recent studies suggest that m6A deposition and proteins involved in the m6A pathway play a diverse set of roles in either restricting or modulating the lifecycles of select viruses. Here, we report that m6A levels are significantly increased in cells infected with the oncogenic human DNA virus Kaposi’s sarcoma-associated herpesvirus (KSHV). Transcriptome-wide m6A-sequencing of the KSHV-positive renal carcinoma cell line iSLK.219 during lytic reactivation revealed the presence of m6A across multiple kinetic classes of viral transcripts, and a concomitant decrease in m6A levels across much of the host transcriptome.

ORGANISM(S): Homo sapiens

PROVIDER: GSE104621 | GEO | 2018/03/29

REPOSITORIES: GEO

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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.

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N6-methyladenosine modification in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection [MeRIP-seq]

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