Tombusviruses Target a Major Crossroad in the Endocytic and Recycling Pathways via Co-opting Rab7 Small GTPase.

Abstract

Positive-strand RNA viruses induce the biogenesis of unique membranous organelles called viral replication organelles (VROs), which perform virus replication in infected cells. Tombusviruses have been shown to rewire cellular trafficking and metabolic pathways, remodel host membranes, and recruit multiple host factors to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely related carnation Italian ringspot virus (CIRV) usurp Rab7 small GTPase to facilitate building VROs in the surrogate host yeast and in plants. Depletion of Rab7 small GTPase, which is needed for late endosome and retromer biogenesis, strongly inhibits TBSV and CIRV replication in yeast and in planta. The viral p33 replication protein interacts with Rab7 small GTPase, which results in the relocalization of Rab7 into the large VROs. Similar to the depletion of Rab7, the deletion of either MON1 or CCZ1 heterodimeric GEFs (guanine nucleotide exchange factors) of Rab7 inhibited TBSV RNA replication in yeast. This suggests that the activated Rab7 has proviral functions. We show that the proviral function of Rab7 is to facilitate the recruitment of the retromer complex and the endosomal sorting nexin-BAR proteins into VROs. We demonstrate that TBSV p33-driven retargeting of Rab7 into VROs results in the delivery of several retromer cargos with proviral functions. These proteins include lipid enzymes, such as Vps34 PI3K (phosphatidylinositol 3-kinase), PI4Kα-like Stt4 phosphatidylinositol 4-kinase, and Psd2 phosphatidylserine decarboxylase. In summary, based on these and previous findings, we propose that subversion of Rab7 into VROs allows tombusviruses to reroute endocytic and recycling trafficking to support virus replication. IMPORTANCE The replication of positive-strand RNA viruses depends on the biogenesis of viral replication organelles (VROs). However, the formation of membranous VROs is not well understood yet. Using tombusviruses and the model host yeast, we discovered that the endosomal Rab7 small GTPase is critical for the formation of VROs. Interaction between Rab7 and the TBSV p33 replication protein leads to the recruitment of Rab7 into VROs. TBSV-driven usurping of Rab7 has proviral functions through facilitating the delivery of the co-opted retromer complex, sorting nexin-BAR proteins, and lipid enzymes into VROs to create an optimal milieu for virus replication. These results open up the possibility that controlling cellular Rab7 activities in infected cells could be a target for new antiviral strategies.