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

The experiment was performed three times by swapping the labelled derivatives of 123 arginine and lysine as described in the materials and methods. Silver stain of the 124 purified complexes confirmed the presence of the bait proteins, with both the 125 uncleaved and cleaved forms of NS1/2 and NS2 being highly enriched ( Fig 2B) . As 126 expected, complexes purified from NS1/2-Flag virus infected cells co-purified 127 untagged NS4 and vice versa (Fig 2B) , as we have previously shown these proteins 128 to interact to form a complex (Thorne et al., 2012) . Western blot analysis of the 129 purified complexes confirmed that viral non-structural and structural proteins were 130 specifically enriched in the purified complexes, including NS5 (VPg)-containing 131 8 proteins identified using NS1/2 and NS4 (Spearman correlation of 0.8832), fitting 140 with our prior knowledge that both proteins form a complex during viral replication 141 (Thorne et al., 2012) . Ontology analysis indicated that proteins involved in vesicle 142 transport and fatty acid metabolism were significantly enriched ( Fig S2 and Table 143 S3), fitting with previous observations that the viral replication complex is associated 144 with cytoplasmic membranous structures (Cotton et al., 2017; Hyde and Mackenzie, 145 2010; Hyde et al., 2009) . Several host proteins previously identified in a variety of 146 biochemical and genetic screens were enriched ( Fig S2 and Table S3 ) providing 147 additional confidence that the approach identified biologically relevant interactions. 148