Uncovering viral RNA-host cell interactions on a proteome-wide scale

Abstract

RNA viruses interact with a wide range of cellular RNA-binding proteins (RBPs) during their life cycle. The prevalence of these host-virus interactions has been highlighted by new methods that elucidate the composition of viral ribonucleoproteins (vRNPs). Applied to 11 viruses so far, these approaches have revealed hundreds of cellular RBPs that interact with viral (v)RNA in infected cells. However, consistency across methods is limited, raising questions about methodological considerations when designing and interpreting these studies. Here, we discuss these caveats and, through comparing available vRNA interactomes, describe RBPs that are consistently identified as vRNP components and outline their potential roles in infection. In summary, these novel approaches have uncovered a new universe of host-virus interactions holding great therapeutic potential.

Keywords: RNA; RNA-binding proteins; proteomics; viral ribonucleoprotein; virus; virus–host interactions.

Figure 1

Approaches to elucidate the viral RNA (vRNA) interactome. (A) Schematic representation of the critical steps of the methods to elucidate vRNA–protein interactions and the alternatives for each step. The name and workflow of each approach are detailed in (B). Abbreviations: 4SU, 4-thiouridine; ActD, actinomycin D; ChIRP-MS, comprehensive identification of RNA-binding proteins by mass spectrometry; CLAMP, crosslink-assisted messenger ribonucleoprotein purification; Fvo, flavopiridol; HyPR-MS, hybridization purification of RNA–protein complexes followed by mass spectrometry; mRNP, messenger ribonucleoprotein; RAP-MS, RNA antisense purification and quantitative mass spectrometry; SPRI, solid-phase reversible immobilisation; TUX-MS, thiouracil cross-linking mass spectrometry; VIR-CLASP, viral cross-linking and solid-phase purification; vRIC, viral RNA interactome capture. See also [25., 26., 27.,40., 41., 42., 43., 44., 45.,50., 51., 52., 53.].

Figure I

Sources of ‘noise’ in a viral (v)RNA interactome experiment. (A–G) Schematic representation of contaminants that may affect the different vRNA interactome approaches.

Figure 2

Defining the core viral RNA (vRNA) interactome. (A–C) Venn diagrams comparing the vRNA interactome datasets for the three viral families with multiple available datasets: (A) Togaviridae [,; W. Kamel, unpublished], (B) Coronaviridae [26,43,45,46], and (C) Flaviviridae [42,44,51]. Only datasets with at least two biological replicates were included. Cell type and capture method are listed under virus name. (D) Venn diagram comparing the Togaviridae, Coronaviridae, and Flaviviridae supersets. The overlap (195 proteins) between them is referred to here as the ‘core viral interactome.’ (E) Density plot showing the occurrence of proteins included and excluded in the core vRNA interactome in individual vRNA interactome datasets. (F) Proportion of the core vRNA interactome that has been linked to viral infection, either through gene ontology (GO) annotation or in the literature. PubMed was searched using the R package Rismed with the search terms [(protein) AND (virus OR viral)]. Any linked to viruses six or more times was classified as virus-linked. GO term annotation was extracted using the R package biomaRt. (G) Proportion of proteins in the vRNA interactome that are annotated with the GO term ‘RNA binding.’ (H) Proportion of proteins with classical RNA-binding domains (RBDs), nonclassical RBDs, and no known RBDs. This is shown for the core vRNA interactome, as well as its components that have been linked to infection and those without links (panel f). Individual protein-level information relating to panels f, g, and h is available in Supplementary Table 3. Abbreviations: CHIKV, Chikungunya virus; ChIRP-MS, comprehensive identification of RNA-binding proteins by mass spectrometry; CLAMP, crosslink-assisted messenger ribonucleoprotein purification; DENV, dengue virus; RAP-MS, RNA antisense purification and quantitative mass spectrometry; RBD, RNA-binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SINV, Sindbis virus; TUX-MS, thiouracil cross-linking mass spectrometry; vRIC, viral RNA interactome capture; ZIKV, Zika virus.

Figure 3

Cellular RNA-binding proteins (RBPs) participate in virtually all stages of viral infection. Schematic representation of the viral life cycle, highlighting the processes involved in viral (v)RNA metabolism. The members of the core vRNA interactome involved in these processes are highlighted in the coloured boxes. RBPs can regulate vRNA fate; however, emerging data suggest that vRNA may, in some instances, regulate protein function, as outlined in the grey box. This alternative regulatory process is referred to as ‘riboregulation.’ RNP, ribonucleoprotein; ssRNA, single-stranded RNA.