Rotavirus NSP1 subverts the antiviral oligoadenylate synthetase-RNase L pathway by inducing RNase L degradation

The interferon (IFN)-inducible 2′,5′-oligoadenylate synthetase (OAS)-RNase L pathway plays a critical role in antiviral immunity. Group A rotaviruses, including the simian SA11 strain, inhibit this pathway through two activities: an E3-ligase related activity of NSP1 that degrades proteins necessary for IFN signaling, and a phosphodiesterase (PDE) activity of VP3 that hydrolyzes the RNase L-activator 2′,5′-oligoadenylate. Unexpectedly, we found that a recombinant (r) SA11 double mutant virus deficient in both activities (rSA11-VP3H797R-NSP1ΔC17) retained the ability to prevent RNase L activation. Mass spectrometry led to the discovery that NSP1 interacts with RNase L in rSA11-infected HT29 cells. This interaction was confirmed through copulldown assay of cells transiently expressing NSP1 and RNase L. Immunoblot analysis showed that infection with wild-type rSA11 virus, rSA11-VP3H797R-NSP1ΔC17 double mutant virus, or single mutant forms of the latter virus all resulted in the depletion o..., General methods are provided in doi: https://journals.asm.org/doi/10.1128/mbio.02995-22
Detailed methods for analyzing the interactome of NSP1 are provided below.
Protein digestion
Individual samples containing proteins bound to magnetic beads were denatured in 8 M urea in 100 mM ammonium bicarbonate. Samples were incubated for 45 min at 57 °C with 10 mM Tris(2-carboxyethyl)phosphine hydrochloride to reduce cysteine residue side chains. These side chains were then alkylated with 20 mM iodoacetamide for one hour in the dark at 21 °C. The urea was diluted to 1 M urea using 100 mM ammonium bicarbonate. Trypsin was added at 1:100 w/w and the samples were placed on a rotator and digested for 14 hours at 37 °C.
Mass spectrometry (MS)
The beads were spun down an immobilized against the wall of the microcentrifuge tube using a magnet. The supernatant was removed and desalted using ZipTip pipette tips (EMD Millipore), dried down and resuspended in 0.1% formic acid. Peptides were analyzed by LC-M...,