Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation

Myra Hosmillo; Jia Lu; Michael R. McAllaster; James B. Eaglesham; Xinjie Wang; Edward Emmott; Patricia Domingues; Yasmin Chaudhry; Timothy J Fitzmaurice; Matthew K.H. Tung; Marc Panas; Gerald McInerney; Nicholas Locker; Craig B. Willen; Ian Goodfellow

Laboratories). BV-2 cells were maintained in SILAC medium for 2 weeks to ensure 798 complete metabolic labelling of proteins. Labelling of HEK-293T cells was performed 799 essentially as described for BV-2 cells, with the omission of 10 mM HEPES and 1X 800 non-essential amino acids from the cell culture media. 801 802 DNA based recovery of murine norovirus. Experiments were performed according 803 to previously published protocols (Chaudhry et al., 2007) . Briefly, BSRT7 cells were 804 infected to an MOI of 0.5-1 PFU/cell with fowlpox virus expressing T7 RNA 805 polymerase. Cells were then transfected with a plasmid encoding the MNV full length 806 clone, or a derivative thereof (e.g. pT7 MNV 383FLAG 3'Rz or pT7 MNV 2600FLAG 807 3'Rz, our FLAG-tagged virus constructs containing FLAG tags in either NS1/2 or 808 NS4 respectively). MNV was harvested by freeze-thaw at 24h post-transfection. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/571455 doi: bioRxiv preprint 37 FLAG and GFP-TRAP immunoprecipitation. FLAG immunoprecipitations were 845 performed following the manufacturer's protocol (FLAG M2 beads, Sigma Aldrich) as 846 described (Thorne et al., 2012) . In brief, protein concentration in lysates was 847 normalized using BCA. Lysates were then diluted with 1 volume of wash buffer (10 848 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA). Equal volumes of anti-FLAG 849 affinity gel were dispensed into either WT infected cell lysates, or lysates of cells 850 infected with NS1/2-FLAG or NS4-FLAG. Binding was carried out overnight at 4 o C 851 with rotation. After binding, beads were washed 3 times with wash buffer. All liquid 852 was carefully removed from each tube, before boiling in SDS-PAGE loading buffer 853 for 10 minutes. GFP-trap immunoprecipitation of GFP-tagged VPg was 854 accomplished using GFP-trap beads (Chromotek) per the manufacturer's protocol, 855 as described . RNase cocktail (Ambion) was also 856 included in the lysis buffer at a concentration of 5 μl/ml to prevent non-specific 857 interactions mediated by RNA. In all cases, light, medium, and heavy-labelled 858 proteins eluted from the beads for each experimental replicate were pooled together 859 in a ratio of 1:1:1 before submission for mass spectrometry analysis at the University 860 of Bristol Proteomics Facility. 861 862 Mass spectrometry analysis. Mass spectrometry analysis was performed at the 863 University of Bristol Proteomics Facility. In brief, samples were run into precast SDS-864 PAGE gels for 5 minutes, the entire sample cut from the gel as a single band, and 865 then subjected to in-gel tryptic digestion including reduction and alkylation using a 866