Mammalian Orthoreovirus Factories Modulate Stress

21 Mammalian orthoreovirus (MRV) infection induces phosphorylation of translation initiation 22 factor eIF2α which promotes formation of discrete cytoplasmic inclusions, termed stress 23 granules (SGs). SGs are emerging as a component of the innate immune response to virus 24 infection, and modulation of SG assembly is a common mechanism employed by viruses to 25 counter this antiviral response. We previously showed that MRV infection induces SGs early, 26 then interferes with SG formation as infection proceeds. In this work, we found that SG 27 associated proteins localized to the periphery of virus-encoded cytoplasmic structures, termed 28 virus factories (VFs), where viral transcription, translation, and replication occur. The 29 localization of SG proteins to VFs was dependent on polysome dissociation and occurred via 30 association of SG effector protein, G3BP1, with MRV non-structural protein σNS, which 31 localizes to VFs via association with VF nucleating protein, μNS. Deletion analysis of the σNS 32 RNA binding domain and G3BP1 RNA (RRM) and ribosomal (RGG) binding domains showed 33 that the association and VF localization of G3BP1 is not occurring solely through RNA or 34 ribosomal binding, but requires both RNA and ribosomal binding domains of G3BP1 for 35 maximal VFL localization and σNS association. Co-expression of σNS and μNS resulted in 36 disruption of normal SG puncta, and in cells lacking G3BP1, MRV replication was enhanced in a 37 manner correlating with strain-dependent induction of host translation shutoff. These results 38 suggest that σNS association with and relocalization of G3BP1 to the VF periphery plays a role 39 in SG disruption to facilitate MRV replication in the host translational shutoff environment. 40