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Mapping viral protease-mediated processing of astrovirus nonstructural polyprotein C4PR_LIV

ABSTRACT: Many viruses with compact RNA genomes use a replication strategy involving the translation and proteolytic processing of a polyprotein. Astroviruses include a range of human and animal species and can infect cells of intestinal and neuronal origin. These viruses have positive-sense RNA genomes and translate their nonstructural polyprotein, which is subsequently processed by viral and cellular proteases. The polyprotein cleavage sites are predicted but not experimentally confirmed. Using N-terminomics and a complementary range of other experimental techniques, we mapped polyprotein cleavage sites in classical human astrovirus 1 (HAstV1) and neurotropic astrovirus strain MLB2, providing a map of individual proteins and their boundaries. Furthermore, we characterize processing precursors and replication properties of processing-defective mutant replicons, further elucidating the importance of polyprotein cleavage in astroviruses. Understanding polyprotein processing dynamics is essential for interpreting viral infection stages and identifying new drug targets and antiviral strategies.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Human Astrovirus 1 Human Astrovirus Mlb2

SUBMITTER: Leandro Xavier Neves

LAB HEAD: Ed Emmott

PROVIDER: PXD058484 | Pride | 2026-06-07

REPOSITORIES: Pride

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			Action	DRS
	2023-07-14-decoys-reviewed-contam-UP000005640-astrovirus_sixframetranslation.fas	Other
	LN231114_pAVIC_MLB2_chymotrypsin_Nterm_bRP1.mzML	Mzml
	LN231114_pAVIC_MLB2_chymotrypsin_Nterm_bRP1.raw	Raw
	LN231114_pAVIC_MLB2_chymotrypsin_Nterm_bRP2.mzML	Mzml
	LN231114_pAVIC_MLB2_chymotrypsin_Nterm_bRP2.raw	Raw

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Project description:Glands were frozen at -80 ºC immediately after being retrieved and stored under this condition until initiating the proteomics analysis protocol. Glands were washed with cold PBS and immersed in 120 l of homogenization buffer (50 mM Tris-HCl, pH 6.8, 10 mM DTT, 4% (w/v) SDS). Glands were boiled for 5 min, incubated overnight at 4 ºC and centrifuged at 4 ºC and 13000 rpm for 10 min. The whole gland proteome of each gland was concentrated as described elsewhere (Bonzon-Kulichenko, F. Garcia-Marques, M. Trevisan-Herraz and J. Vazquez, Journal of Proteome Research, 2015, 14, 700-710 (DOI:10.1021/pr5007284).Briefly, samples were loaded into an SDS-PAGE gel (0.5 mm-thick, 4% stacking, and 10% resolving) and the electrophoresis process was stopped when the front entered 2 mm into the resolving gel. The unseparated protein bands were then visualized by Coomassie staining, excised and digested at 37 ºC overnight with 500 l of 25 g/l chymotrypsin (Promega) in 100 mM Tris-HCl, pH 7.8 containing 10 mM CaCl2. The cleaved peptides were extracted by incubation for 2 h in 100 mM Tris-HCl, pH 7.8 on shaking, acidized with 1% (v/v) trifluoroacetic acid, desalted onto C18 cartridges (Oasis, Waters Corporation, Milford, MA, USA), and dried down.The analysis of the samples proceeded through liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Easy nLC 1000 nano-HPLC coupled to a Q-Exactive Orbitrap mass spectrometer (Themo Scientific). Peptides were injected onto a C18 reverse-phase precolumn (Acclaim PepMap100, 75-m i.d., 3-m particle size, 2-cm length, Thermo Scientific), and a reverse-phase analytical column (Acclaim PepMap 100, 75-m, i.d., 3-m particle size, 50-cm length, Thermo Scientific) in buffer A [0.1% formic acid (v/v)] and eluted with a 60 min linear gradient of buffer B [90% acetonitrile, 0.1% formic acid (v/v)], at 200 nL/min. Mass spectrometry (MS) runs consisted of 70000 FT-resolution spectra in the 390-1200 m/z wide range, followed by data-dependent MS/MS spectra of the 15 most intense parent ions acquired along the chromatographic run. High-energy collisional (HCD) fragmentation was performed at 27% of normalized collision energy and the isolation window for the parent ion mass was established at 2 Da.

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Mapping viral protease-mediated processing of astrovirus nonstructural polyprotein C4PR_LIV

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