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

In addition, to minimise the impact of gRNAs that may have deleterious effects on 164 long term cell viability and to increase our ability to detect genes that may be 165 important, but not essential, for norovirus-induced cell death, the infection was 166 reduced to 24 hours as compared to 2-10 days post infection in previous studies. 167 BV-2-Cas9 expressing cells were infected with lentiviruses carrying the Brie gRNA 168 library carrying 78,637 independent guide RNAs to 19,674 genes (Doench et al., 169 2016) . The transduced cells were then infected with two MNV strains, CW3 and 170 CR6, which cause acute and persistent infections in immunocompetent mice 171 respectively (Nice et al., 2012; Thackray et al., 2007) , and guide RNA abundance 172 compared to mock infected cells at 24 hours post infection as illustrated in Fig 3A. 173 being negatively selected (Fig 3B) , whereas for MNV-CW3 279 and 19 genes were 179 positively and negatively selected respectively ( Fig 3B) . In most cases, there was a 180 clear correlation between the datasets obtained using either strain ( Fig 3C) . STARS 181 analysis was used to ranks genes with positive and negative values (Table S4 ). In 182 both screens, the MNV receptor Cd300lf was the most highly positively selected 183 gene identified, in agreement with previous reports (Haga et al., 2016; Orchard et al., 184 2016) . The second most highly enriched gene was G3BP1, a gene also identified in 185 one of the two previous CRISPR screens performed on norovirus infected cells 186 . 187 10 A comparison of the data obtained from all three approaches allowed us to identify 189 several host proteins that were common to all screens (Table S5) . G3BP1, the core 190 stress granule component was identified in all three screens as a potential host 191 factor essential for norovirus infection. G3BP1 was found to be associated with the 192 MNV and NV VPg proteins (Fig 1D) , enriched in viral replication complexes purified 193 using either NS1/2 or NS4 flag tagged viruses ( Fig 2D) and identified in a CRISPR 194 screen using two different MNV strains as a putative pro-viral factor involved in the 195 norovirus life cycle ( Fig 3C) . 196