Mammalian Orthoreovirus Factories Modulate Stress Granule Protein Localization by Interaction with G3BP1

Abstract

Mammalian orthoreovirus (MRV) infection induces phosphorylation of translation initiation factor eIF2 which promotes formation of discrete cytoplasmic inclusions, termed stress granules (SGs). SGs are emerging as a component of the innate immune response to virus infection, and modulation of SG assembly is a common mechanism employed by viruses to counter this antiviral response. We previously showed that MRV infection induces SGs early, then interferes with SG formation as infection proceeds. In this work, we found that SG associated proteins localized to the periphery of virus-encoded cytoplasmic structures, termed virus factories (VFs), where viral transcription, translation, and replication occur. The localization of SG proteins to VFs was dependent on polysome dissociation and occurred via association of SG effector protein, G3BP1, with MRV non-structural protein {sigma}NS, which localizes to VFs via association with VF nucleating protein, NS. Deletion analysis of the {sigma}NS RNA binding domain and G3BP1 RNA (RRM) and ribosomal (RGG) binding domains showed that the association and VF localization of G3BP1 is not occurring solely through RNA or ribosomal binding, but requires both RNA and ribosomal binding domains of G3BP1 for maximal VFL localization and {sigma}NS association. Co-expression of {sigma}NS and NS resulted in disruption of normal SG puncta, and in cells lacking G3BP1, MRV replication was enhanced in a manner correlating with strain-dependent induction of host translation shutoff. These results suggest that {sigma}NS association with and relocalization of G3BP1 to the VF periphery plays a role in SG disruption to facilitate MRV replication in the host translational shutoff environment.\n\nIMPORTANCESGs and SG effector proteins have emerged as important, yet poorly understood, players in the hosts innate immune response to virus infection. MRV infection induces SGs early during infection that are dispersed and/or prevented from forming during late stages of infection despite continued activation of the eIF2 signaling pathway. Cellular and viral components involved in disruption of SGs during late stages of MRV infection remain to be elucidated. This work provides evidence that MRV disruption of SGs may be facilitated by association of MRV non-structural protein {sigma}NS with major SG effector protein G3BP1 and subsequent localization of G3BP1 and other SG associated proteins around the periphery of virus encoded factories, interrupting the normal formation of SGs. Our findings also reveal the importance of G3BP1 as an inhibitor of MRV replication during infection for the first time.