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

and well-formed colonies were fixed in 10% formaldehyde and stained with toluidine 923 blue. A similar protocol was followed to assess colony formation in U2OS cells with 924 the exception that selections were maintained for up to 12 days post transfection. from MNV-1 virus particles was transfected in BV-2 cells using NEON™ as 956 previously described (Yunus et al., 2010) . Total cell lysates were harvested at 3 and 957 9 hours post transfection with RIPA buffer. 10μg total lysates were analysed by 4-958 12% SDS-PAGE (Invitrogen) and antibodies against MNV, VPg, G3BP1 and GAPDH 959 were used for detection using LI-COR® Odyssey® CLx. Virus yield was determined 960 by TCID 50 . For strand-specific qPCR detection of MNV RNA, total cellular RNA was 961 extracted using GeneElute Mammalian Total RNA Miniprep kit (Sigma). RT-qPCR 962 was performed as described previously (Vashist et al., 2012a) . The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. Preparation of BV2 S10 cytoplasmic extracts. Preparation of BV-2 S10 extracts 976 was based on a previously published protocol (Rakotondrafara and Hentze, 2011; 977 2006) . BV-2 cells were harvested, washed with PBS, and lysed with 1x packed 978 volume of hypotonic buffer containing 10 mM HEPES pH7.6, 10 mM potassium 979 acetate, 0.5 mM magnesium acetate, 5 mM DTT, 1x protease inhibitors cocktail 980 (EDTA-free, Roche). Cells were lysed on ice for 45 minutes, then passed through 981 25G and 27G needles to achieve >95% lysis. Cell lysates were then centrifuged at 982 The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 Figure S11