Controlling the lytic switch; can m6A-modified RNA be used as an Anti-Viral Target?

Abstract

KSHV has a biphasic life cycle encompassing a latent state and lytic replication. The KSHV replication and transcription activator viral protein, encoded from open reading frame 50 (ORF50), is the key viral protein which drives the switch between the latent and lytic phases (Guito and Lukac, 2012). We have recently demonstrated that KSHV manipulates the host cell N6-methyl adenosine (m6A) RNA modification pathway to enhance viral gene expression. Specifically, we have shown that the KSHV ORF50 transcript is m6A methylated, allowing the recruitment of the m6A reader protein, Staphylococcal nuclease domain-containing protein 1 (SND1), resulting in the stabilisation of the ORF50 transcript and efficient KSHV lytic replication (Baquero-Perez et al. 2019).

Further analysis of the m6A modified site with the ORF50 transcript has identified an RNA stem-loop, termed ORF50-1, which is a m6A-modified 43-mer, essential for SND-1 binding, thought to occur in a secondary structure/ sequence-dependent manner.

Generating in silico 3D structures of the ORF50-1 RNA in its native ‘N’ and m6A-modified ‘M’ form confirms that the presence of the m6A-modification has repercussions in the lower part of the stem-loop and predicts a different secondary structure(collab. Pasquale and Roeder).

Due to the obvious structural differences anticipated, RNA-binding ligands have been identified using Small Molecule Microarrays (SMMS) (collab. Fullenkamp and Schneekloth). By investigating these ligands in biophysicalexperiments, we have verified RNA-binding and optimised cell-based assays to assess anti-viral properties. It is hoped these experiments will highlight the importance of A versus m6A within the lytic phase of KSHV’s lifecycle.