Defining Distinct RNA-Protein Interactomes of SARS-CoV-2 Genomic and Subgenomic RNAs

Abstract

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. Individual interactomes indicated viral associations with cell response pathways, including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We tested the significance of three protein interactors in these pathways (APOBEC3F, PPP1CC, and MSI2) using siRNA knockdowns, with several knockdowns affecting viral gene expression, most consistently PPP1CC. This study describes a new technology for high-resolution studies of SARS-CoV-2 RNA regulation and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

Identification of Host Proteins Involved in Hepatitis B Virus Genome Packaging

“…pgRNA capture efficiencies averaged 49 and 57% for LJ144 and EL43 transfected cells, respectively, and pgRNA was enriched by a factor of 456 and 253 compared to the scrambled control for LJ144 and EL43, respectively (Figure C,D and Supporting Table 2). These data were consistent with previous HyPR-MS experiments and demonstrated the high degree of specificity necessary for mass spectrometry and comparative protein analysis. ,, …”

“…One strategy to further investigate the role of host proteins in viral RNA packaging is RNA-protein interaction analysis such as hybridization purification of RNA-protein complexes followed by mass spectrometry (HyPR-MS). − In HyPR-MS, cells are cross-linked with formaldehyde to stabilize RNA-protein complexes, and the complexes are then specifically captured by hybridization to biotin-conjugated complementary oligonucleotides. The interacting proteins are then identified and quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS).…”