Emerging RNA-centric technologies to probe RNA-protein interactions: importance in decoding the life cycle of positive sense single strand RNA viruses and antiviral discovery

Figures

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  Figure 1. Recognition of +ssRNA viruses by the host innate immune pathways and generation of antiviral response. RIG-I and MDA5 recognize dsRNA, TLR3 and TLR7/8 sense dsRNA and ssRNA, respectively, and activate the indicated pathways to express type I and type III interferons and proinflammatory cytokines. dsRNA also activates RNase L, which cleaves the former. Viral proteins can damage mitochondria and/or activate the inflammasome. RIG-I, Retinoic acid-inducible gene; MDA5, Melanoma differentiation-associated gene 5; LGP2, Laboratory of genetics and physiology 2; MAVS, Mitochondrial antiviral signaling protein; TRAF3, TNF receptor associated factor 3; TBK1, TANK-binding kinase 1; IKKϵ, IκB kinase ϵ; IRF3/7, Interferon regulatory factor 3 or 7; TLR3, toll-like receptor 3; TLR7/8, toll-like receptor 7 or 8; TRIF, TIR-domain containing adaptor inducing interferon-β; RIP-1, receptor-interacting protein 1; TRAF6, TNF receptor associated factor 6; TAK1, TGFβ-activated kinase 1; IKKα/β, IκB kinase α/β; MyD88, Myeloid differentiation primary response 88; IRAK1,4, interleukin-1 receptor-associated kinase 1,4; OAS, oligoadenylate synthetase; 2’-5’ A, 2’-5’ oligoadenylate; NLRP3, NOD-like receptor…

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  Figure 2. Simplified illustration of life cycle of +ssRNA viruses. The RNA virus life cycle has four major steps- entry, replication, assembly, and egress. After entry into the host and uncoating of the viral capsid, viral genomic RNA is translated to produce the non-structural polyprotein (NSP), which is subsequently processed into individual subunits. Viral RdRp assembles a RNA-protein complex, which interacts with the RNA-protein complexes assembled at 5’- and 3’- termini of the viral genomic RNA to form the viral replication complex. Viral genomic RNA likely forms a closed loop structure during replication. Antisense strand (-) as well as sub-genomic (sg) and genomic (g) RNA strands are synthesized by replication. Sub-genomic RNA is translated to produce the structural proteins (SP) that assembles the viral capsid, which encapsulates the genomic RNA. Progeny virions are subsequently released outside. A B C D illustrate 5’-UTR-interacting host proteins; a b c d illustrate 3’-UTR-interacting host proteins; h* illustrate host proteins interacting with the RdRp-bound RNA-protein complex.

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  Figure 3. Schematic of the RNA regulatory elements present in the genome of HCV and SARS-CoV-2. Stem loops in the 5’ and 3’ UTR (untranslated region) regions have been indicated in black color. Stem loops present in internal region are represented against corresponding proteins. Top schematic is for Hepatitis C virus which includes - C, Core/capsid protein; E1 and E2, Envelope glycoproteins; p7, Viroporin; NS, non-structural proteins. The bottom schematic is for SARS-CoV-2 which includes – ORF 1a to ORF14, Open reading frame; S, Spike protein; E, Envelope protein; M, Membrane protein; N, Nucleocapsid protein. S, M, E, N are the structural proteins.

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  Figure 4. In vitro and in vivo methods to detect RNA-Protein interactions in +ssRNA viruses. RaPID, RNA-Protein Interaction Detection Assay; RNA BioID- RNA proximity biotinylation; incPRINT- In-cell protein-RNA interaction; MS2-BioTRAP, MS2-in vivo Biotin Tagged RNA Affinity Purification; MTRAP-MS, MS2-tagged RNA affinity purification and Mass spectrometry; RAP-MS, RNA Antisense Purification followed by Mass Spectrometry; iDRiP, Identification of Direct RNA-interacting Proteins; TRIP, Tandem RNA Isolation Procedure; PAIR, Peptide-Nucleic acid Assisted Identification followed by Mass Spectrometry; vRIC-MS, Viral RNA Interactome Capture followed by Mass Spectrometry; VIR-CLASP, Viral Cross-linking And Solid-phase Purification; CHART-MS, Capture Hybridization Analysis of RNA Targets followed by Mass Spectrometry; ChIRP-MS, Comprehensive identification of RNA-binding proteins by mass spectrometry.

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  Figure 5. Schematic of in vitro methods to detect RNA-protein interactions. (A) In-vitro pull down assay. (B) Microarray-based binding assay.

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  Figure 6. Non-crosslinked in vivo methodologies to study RNA-protein interactions. (A) Yeast three hybrid (Y3H) assay, (B) RaPID assay and RNA BioID, (C) incPRINT, (D) dCas13-based technique.

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  Figure 7. Schematic of major steps in the in vivo experimental methods involving mass spectrometry analysis to study viral RNA-protein interactions. White circle indicates formaldehyde crosslinking, white and Blue star indicate UV crosslinking, yellow circle indicates biotin tag.

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  Figure 8. Schematic of computational methods to study RNA-protein interactions.