Structural and biophysical studies of RNA-protein complexes that stimulate ribosomal frameshifting during viral infection

Background

Programmed ribosomal frameshifting (PRF) is a common viral translational control strategy in which elongating ribosomes 'slip' into a different reading frame when they encounter a structured 'stimulatory element' in the viral RNA that is difficult to unwind. PRF usually occurs at a fixed efficiency, thus specifying the relative stoichiometry of gene products encoded in the two reading frames. In conventional PRF, the stimulatory element is a pseudoknot (e.g. SARS-CoV-2) or stem-loop (e.g. HIV-1). However, we have recently discovered that in some viruses, an RNA-protein complex is required. In cardioviruses (e.g. EMCV) -1 PRF only occurs when the virally-encoded 2A protein recognises its cognate RNA element (PMID: 31180502). In arteriviruses (e.g. PRRSV), -2 PRF is stimulated by a complex between viral protein nsp1b, host poly(C) binding protein, and a C-rich stretch in the RNA (PMID: 27257056).

Stimulatory elements present an intriguing paradox - they must be stable enough to present a blockade to the elongating ribosome, yet also dynamic and plastic enough to allow remodelling and unwinding as translation continues. Recent studies suggest that they may exist in several conformations, and that protein components (e.g. 2A) also bind to the ribosome itself. Furthermore, several host anti-viral proteins have recently been identified (e.g. Shiftless, ZAP-1) that bind to and modulate the stability of these elements (PMID:34202160). However, a lack of structural data on these important RNA-protein complexes has hampered our understanding.

Objectives

To define and purify minimal RNA-protein complexes and determine their structure, thus defining the "rules" governing the molecular interaction.

To characterise the stability and conformational dynamics of these elements, and examine how anti-viral host proteins affect these properties.

To investigate specific interactions between stimulatory elements and ribosomes

Novelty

The only structures of mammalian PRF stimulatory elements are the HIV stem-loop and SARS-CoV-2 pseudoknot. No structural data exist for RNA-protein complexes. The mechanistic basis for the action of anti-viral proteins is unknown.

Timeliness

This builds on a recent body of work from the primary supervisor characterising the cardiovirus system (https://www.biorxiv.org/content/10.1101/2020.08.11.245035v2) and a preliminary analysis of the Shiftless anti-viral factor (PMID:34202160). Recent improvements in cryo-EM now enable structure determination of small structures RNAs and their complexes (e.g. PMID: 34426697)

Experimental Approach

Protein, RNA and ribosome subunit purification, complex reconstitution

Fluorescent labelling of RNA and protein molecules

SEC-MALLS, ITC, SPR, MST and FCS for affinity and stoichiometry

Structure determination of complexes by X-ray crystallography and cryo-EM

FRET to study transitions between RNA conformers; unfolding and refolding kinetics

Optical tweezers to measure stability/force required for unwinding