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Next Generation Sequencing analysis of KSHV lytic replication in TREx-BCBL1-RTA cells

ABSTRACT: Purpose: miR-Seq was utilised to identify miRNAs which are altered during the course of KSHV lytic replication at 0, 16 and 24 hours post reactivation in TREx-BCBL1-RTA cells. Methods: Virus lytic replication was induced via addition of 2 µg/mL doxycycline hyclate (Sigma-Aldrich). Total RNA was extracted from TREx-BCBL-1s at 0, 16 and 24 hours post lytic induction. Small RNA libraries were prepared using the TruSeq Small RNA Library Prep Kit (Illumina). Quality filtered (Q < 20), and adapter trimmed reads (Trimmomatic v0.39) [59] were aligned to the GRCh38/hg38 assembly of the human genome using Bowtie2 (V 2.4.2).

ORGANISM(S): Homo sapiens

PROVIDER: GSE186652 | GEO | 2022/02/09

REPOSITORIES: GEO

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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.

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Next Generation Sequencing analysis of KSHV lytic replication in TREx-BCBL1-RTA cells

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