Ribosomal frameshifting and readthrough in virus gene expression. (360G-Wellcome-092334_Z_10_Z)

This project will focus on the role of recoding in the replication of the model retroviruses Rous sarcoma virus (RSV; frameshifting) and murine leukemia virus (MuLV; readthrough) and the coronavirus murine hepatitis virus (MHV, frameshifting). Specific mutations will be introduced into the recoding signals such that individual viruses, over a broad range, exhibit a defined frameshifting or readthrough efficiency. The replication of these engineered viruses will be assessed and their tolerance to changes in efficiency determined. As an alternative approach, recoding efficiency will be modulated using small molecules, including antibiotics known to affect recoding, and by targeting the stimulatory RNA structures with oligonucleotides. The mechanism of the readthrough process will also be investigated. Unlike frameshifting, we know little about how the stimulatory RNA pseudoknot of MuLV perturbs ribosome function. This will be investigated by biochemical analysis of ribosome-pseudoknot co mplexes, using primer-extension/toeprinting, RNA secondary structure probing and cryo-electron microscopy (cryo-EM).Overall, this project aims to establish the extent to which recoding can be modified before virus replication is compromised, to determine whether recoding is a valid target for antiviral intervention and to clarify the mechanism of readthrough, which may identify new approaches to antiviral therapy.