Project description: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).
Project description:We have mapped m6A sites and endogenous SND1 binding sites in the viral and cellular transcriptome using TREx BCBL1-Rta cells. In addition, we have depleted SND1 in TREx BCBL1-Rta cells and BCBL1 cells and analyzed their RNA expression profile both during latency and lytic replication.
Project description:The epitranscriptomic modification m6A is a ubiquitous feature of the mammalian transcriptome. It modulates mRNA fate and dynamics to exert regulatory control over numerous cellular processes and disease pathways, including viral infection. Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation from the latent phase leads to redistribution of m6A topology upon both viral and cellular mRNAs within infected cells. Here we investigate the role of m6A in cellular transcripts upregulated during KSHV lytic replication. Results show that m6A is crucial for the stability of the GPRC5A mRNA, whose expression is induced by the KSHV latent-lytic switch master regulator, the replication and transcription activator (RTA) protein. Moreover, we demonstrate that GPRC5A is essential for efficient KSHV lytic replication by directly regulating NFκB signalling. Overall, this work highlights the central importance of m6A in modulating cellular gene expression to influence viral infection.
Project description:Effect of expression of wild type KSHV RTA or the activation domain (aa608-651) deleted version of RTA in 293T cells on gene expression profiling.
Project description:RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. LPS/TLR4 engagement enhances rhadinovirus reactivation. We developed the HE-RIT cell line, a latent murine A20 B cell inducible for Flag-RTA expression and murine gammaherpesvirus 68 reactivation. LPS acted as a second stimulus to drive virus reactivation from latency in the context of induced expression of FLAG-RTA. We applied RNAseq to examine for genome-wide changes in viral gene expression in response to doxycycline-induced RTA-FLAG, alone or in combination with LPS.