Viral crosslinking and solid-phase purification enables discovery of ribonucleoprotein complexes on incoming RNA virus genomes

Abstract

The initial interactions between incoming, pre-replicated virion RNA and host protein factors are important in infection and immunity. Yet currently there are no methods to study these crucial events. We established VIR-CLASP (VIRal Cross-Linking And Solid-phase Purification) to identify the primary viral RNA-host protein interactions. First, host cells are infected with 4-thiouridine (4SU)-labeled RNA viruses and irradiated with 365 nm light to crosslink 4SU labeled viral genomes and interacting proteins from host or virus. The cross-linked RNA binding proteins (RBPs) are purified by solid-phase reversible immobilization (SPRI) beads with protein denaturing buffers, and then identified by proteomics. With VIR-CLASP, only the incoming virion RNAs are labeled with 4SU, so cross-linking events specifically occur between proteins and pre replicated virion RNA. Since solid-phase purification under protein-denaturing conditions, rather than sequence-specific nucleic acid purification, is used to pull-down total RNA and cross-linked RBPs, this method facilitates investigation of potentially all RNA viruses, regardless of RNA sequence. Preparation of 4SU-labeled virus takes ~7 days and VIR-CLASP takes 1 day.

Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions

“…For example, mapping physical associations between the host and virus at the level of protein-protein interactions (PPIs) have defined key pathways relevant to infection (Eckhardt et al, 2020). In parallel to efforts that focus on viral proteins, a number of groups have taken an RNA-centric view of the host-viral interface to understand how host cells recognize and respond to the RNA genome (Kim et al, 2020a;Lenarcic et al, 2013;Ooi et al, 2019;Phillips et al, 2016;Viktorovskaya et al, 2016). Finally, genetic screening efforts provide another strategy to discover cellular proteins and pathways that are essential for viral replication or that are part of the host innate immune responses (Puschnik et al, 2017;Schoggins and Rice, 2011).…”

Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions

“…3d ). To cross validate the eCLIP findings that SARS-CoV-2 proteins interact with host cell RNAs, we validated a subset of these proteins as RBPs using crosslinking and solid phase purification (CLASP 26 ), which stringently captures crosslinked protein-RNA interactions due to denaturing wash conditions. HEK293T cells transiently expressing NSP1, NSP2, NSP12, and ORF9c followed by pulldown of total RNA showed enrichment of these proteins ( Fig.…”

Next-generation sequencing: A new avenue to understand viral RNA–protein interactions

“…As a recent development, VIR-CLASP (VIRal Crosslinking And Solid-phase Purification) also presents a method that is designed to investigate the protein components of the viral RNA-cellular RBP interactions ( 162 ). In VIR-CLASP, in a similar fashion to vPAR-CL ( 24 ), cells are 4SU labelled and infected with virus of choice, such as chikungunya virus (CHIKV) ( 162 ). This is followed by UVA crosslinking, and cell lysis.…”