A recent study published in the WIRES journal summarized the importance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ribonucleic acid (RNA)–protein interactomes in the study of RNA viruses.
Various studies have explored the molecular mechanisms of SARS-CoV-2 infections to develop novel therapeutic methods against coronavirus disease 2019 (COVID-19). However, knowledge is lacking regarding the impact of SARS-CoV-2 infection on host messenger RNA (mRNA) transcription.
Several approaches have been developed to capture and detect interactions between SARS-CoV-2 RNA and proteins. A majority of these use the crosslinking of RNA-protein to capture the target RNA. Some protocols that employ this technique are RNA antisense purification and quantitative mass spectrometry (RAP-MS), comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), or hybridization purification of RNA–protein complexes and mass spectrometry (HyPR-MS).
Most of the interactome capture methods begin with the SARS-CoV-2 infection of the model cell line, then the protein-RNA contacts are cross-linked by either ultraviolet (UV) irradiation or chemical treatment. Crosslinking stabilizes the RNA-protein contacts, thus allowing the isolation of the RNA-protein complex formed. Furthermore, all the SARS-CoV-2 RNA–protein interactome analyses shared four primary steps: isolating the RNA–protein complexes from infected cells, purifying the complexes under denaturing conditions, eluting RNA-bound protein followed RNA digestion, and analyzing the viral proteome with MS.
The ChIRP-MS and RAP-MS methods used the antisense probe capture strategy to assess the SARS-CoV-2 RNA-bound proteome. In RAP-MS, the UV irradiation of infected cells was followed by isolation of the RNAs and bound proteins using deoxyribonucleic acid (DNA) oligonucleotides. The specificity and efficiency of the antisense oligonucleotide capture approach depend upon the design and optimization of the probe. While multiple probes result in efficient captures, they can also cause higher noise interventions. On the other hand, fewer probes increase specificity but decrease the accessibility of the region targeted by the probe. Notably, ChIRP-MS employed 108 short probes that were 20 nucleotides (nt)-long while RAP-MS used 90 nt probes and HyPR-MS used two to three probes, each of length 20–30 nt.
The biological relevance of SARS-CoV-2 RNA–protein interactomes
The major outcomes of SARS-CoV-2 RNA–protein interaction studies are the hosts and/or viral proteins that interacted with the viral RNA, namely the genomic RNA and the single guide RNA (sgRNA). For all the studies, the outcomes included RNA binding proteins (RBPs) and factors related to RNA processing. In the RAP-MS method, the captured proteins were enriched in protein groups related to translation, co-translational membrane-targeting proteins, nonsense-mediated decay (NMD), and viral transcription. Another study that employed RAP-MS identified several proteins associated with mRNA processing, stability control, and RNA export.
The majority of the studies also found that 32%,12%, and 5% of the proteins captured were present in at least two, three, and four of the interactome datasets, respectively. The protein interactors shared by at least three of the datasets consisted of factors that were functionally associated with the cellular processes of RNA viruses. Overall, the protein interactors overlapping across the four datasets were mostly related to different cell lines, experimental conditions, and the method of capture employed. The studies also noted that one of the four datasets had unique proteins, all of which had important roles in viral infection.
High-throughput molecular techniques for RNA-interactome studies of other viruses
High-throughput, proteome- and genome-wide technologies can be applied to RNA viruses other than SARS-CoV-2 with sufficient accuracy. Studies also showed the success of the viral cross-linking and solid-phase purification (VIR-CLASP) method in identifying several proteins bound to the chikungunya virus (CHIKV) and influenza A virus (IAV) genomes.
The authors also found unique as well as shared host factors in these genomes that affected innate immunity and controlled infection progression. Further analysis of strain-specific RNA-protein interactions highlighted the mechanisms of de novo virulence.
Moreover, methods associated with protein-protein interaction (PPI) also noted several observations regarding the RNA virus infection mechanism. A study assessed the protein networks of zika virus (ZIKV) infection to study the interactions between the virus and the vector. This study identified several interactors, including proteins that were potentially associated with pro-viral activity in ZIKV infection. Studies regarding host-virus PPI networks for SARS-CoV-2 showed the complex network between the viral and cellular protein and could potentially help in the development of broad-spectrum anti-coronavirus therapies.
Overall, the study summarized that the knowledge of SARS-CoV-2 RNA-protein interactions will facilitate a better understanding of RNA viral infection mechanisms and enhance the therapy design process against future diseases caused by RNA viruses.