DOI: 10.5061/dryad.wpzgmsbr1
title: Data from: Rotavirus NSP1 subverts the antiviral oligoadenylate synthetase-RNase L pathway by inducing RNase L degradation

description: 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 of endogenous RNase L. The loss of RNase L was reversed by
 addition of the neddylation inhibitor MLN4924, but not the proteasome
 inhibitor MG132. Analysis of additional mutant forms of rSA11 showed that
 RNase L degradation no longer occurred when either the N-terminal RING
 domain of NSP1 was mutated or the C-terminal 98 amino acids of NSP1 were
 deleted. The C-terminal RNase L degradation domain is positioned upstream
 and is functionally independent of the NSP1 domain necessary for
 inhibiting IFN expression. Our studies reveal a new role for NSP1 and its
 E3-ligase related activity as an antagonist of RNase L and uncover a novel
 virus-mediated strategy of inhibiting the OAS-RNase L pathway.
description: 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-MS on an
 Orbitrap Fusion Lumos equipped with an Easy NanoLC1200. Buffer A was 0.1%
 formic acid in water. Buffer B was 0.1% formic acid in 80% acetonitrile.
 Peptides were separated on a one-hour gradient from 0% B to 35% B.
 Peptides were fragmented by HCD. Precursor ions were measured in the
 Orbitrap with a resolution of 120,000. Fragment ions were measured in the
 Orbitrap with a resolution of 60,000. MS data
 analysis Data were analyzed using Proteome Discoverer
 (2.5) to interpret and quantify the relative amounts in a label free
 quantification manner. Data was searched against the Homo Sapiens proteome
 downloaded from Uniprot on 7/30/2020. Trypsin was set as the protease with
 up to two missed cleavages allowed. Carbamidomethylation of cysteine
 residues was set as a fixed modification. Oxidation of methionine and
 protein N-terminal acetylation were set as variable modifications. A
 precursor mass tolerance of 10 ppm and a fragment ion quantification
 tolerance of 0.05 Da were used. Data was quantified using the minora
 feature detector node within Proteome Discoverer.