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

To validate the importance of G3BP1 in the norovirus life cycle we generated G3BP1 199 deficient BV-2 cell lines ( Fig 4A) and examined the impact of G3BP1 ablation on 200 MNV infection. Western blotting confirmed the loss of G3BP1 in the three lines 201 tested and we noted that at in some cases, a concomitant increase in G3BP2 202 expression was observed as has been previously noted (Kedersha et al., 2016) . A 203 clear defect was observed in the ability to replicate to produce infectious virus in 204 three independently selected ΔG3BP1 cell lines ( Fig 4B) . This effect was mirrored by 205 an inability to induce cytopathic effect leading to virus-induced cell death ( Fig 4C) . In 206 contrast, the ability of encephalomyocarditis virus (EMCV) to infect and cause cell 207 death was unaffected by the deletion of G3BP1( Fig 4C) . These data confirm that 208 cells lacking G3BP1 are highly resistant to norovirus infection. To determine if the G3BP1 was also essential for HuNoV, we examined the impact 212 of loss of G3BP1 on human norovirus replication in cell culture using the Norwalk 213 virus replicon. To establish the experimental system, we first confirmed that the 214 presence of VPg on the 5' end of the Norwalk RNA was essential for the replication 215 of the replicon RNA and for the capacity to form G418 resistant colonies. 216