A direct RNA-protein interaction atlas of the SARS-CoV-2 RNA in infected human cells

Abstract

SARS-CoV-2 infections pose a global threat to human health and an unprecedented research challenge. Among the most urgent tasks is obtaining a detailed understanding of the molecular interactions that facilitate viral replication or contribute to host defense mechanisms in infected cells. While SARS-CoV-2 co-opts cellular factors for viral translation and genome replication, a comprehensive map of the host cell proteome in direct contact with viral RNA has not been elucidated. Here, we use RNA antisense purification and mass spectrometry (RAP-MS) to obtain an unbiased and quantitative picture of the human proteome that directly binds the SARS-CoV-2 RNA in infected human cells. We discover known host factors required for coronavirus replication, regulators of RNA metabolism and host defense pathways, along with dozens of potential drug targets among direct SARS-CoV-2 binders. We further integrate the SARS-CoV-2 RNA interactome with proteome dynamics induced by viral infection, linking interactome proteins to the emerging biology of SARS-CoV-2 infections. Validating RAP-MS, we show that CNBP, a regulator of proinflammatory cytokines, directly engages the SARS-CoV-2 RNA. Supporting the functional relevance of identified interactors, we show that the interferon-induced protein RYDEN suppresses SARS-CoV-2 ribosomal frameshifting and demonstrate that inhibition of SARS-CoV-2-bound proteins is sufficient to manipulate viral replication. The SARS-CoV-2 RNA interactome provides an unprecedented molecular perspective on SARS-CoV-2 infections and enables the systematic dissection of host dependency factors and host defense strategies, a crucial prerequisite for designing novel therapeutic strategies.

Structural analysis of SARS-CoV-2 genome and predictions of the human interactome

“…We note that the experiments have been carried out 24 hours after infection 73 , which indicates that the protein interaction landscape might have changed with respect to the early events of replication. Yet, the accordance with our calculations indicates participation of elements involved in controlling RNA processing and editing (DDX1, DDX3X) and translation (IGF2BP1 and IGF2BP2), although proteins such as ADAR and XRCC5 were reported to have poorer binding capacity 73 .…”

“…Comparison with protein-RNA interactions detected in the liver cell line HuH7 73 shows agreement with our predictions. We note that the experiments have been carried out 24 hours after infection 73 , which indicates that the protein interaction landscape might have changed with respect to the early events of replication.…”

“…Using the liver cell line HuH7 a recent experimental study by Schmidt et al 73 . identified SARS-CoV-2 RNA associations within the human host 73 . Through the RAP-MS approach, 571…”

Genome-wide bioinformatic analyses predict key host and viral factors in SARS-CoV-2 pathogenesis

“…We aimed at finding RBPs which potentially interact with SARS-CoV-2 genomes in a conserved and specific way. Five of the proteins predicted to be interacting with the viral genome by our pipeline (EIF4B, hnRNPA1, PABPC1, PABPC4, and YBX1) were experimentally shown to bind to SARS-CoV-2 RNA in an infected human liver cell line, based on a recent preprint [65].…”

Genome-wide bioinformatic analyses predict key host and viral factors in SARS-CoV-2 pathogenesis

“…We aimed at finding RBPs which potentially interact with SARS-CoV-2 genomes in a conserved and specific way. Five of the proteins predicted to be interacting with the viral genome by our pipeline (EIF4B, hnRNPA1, PABPC1, PABPC4, and YBX1) were experimentally shown to bind to SARS-CoV-2 RNA in an infected human liver cell line, based on a recent preprint [67].…”