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

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.