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

Patton, John1 ; Yi, Guanghui1 ; Philip, Asha1 ; Dai, Jin1

Research facility: Indiana University Bloomington

Published May 25, 2023 on Dryad. https://doi.org/10.5061/dryad.wpzgmsbr1

Data files

May 25, 2023 version files 146.81 KB

Abstract

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.

Methods

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.

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

Data files

Abstract

Methods

Works referencing this dataset