| Abstract: | The replication of positive-strand RNA viruses occurs in cytoplasmic membrane-bound virus replication complexes (VRCs). Depending on the virus, distinct cellular organelles such as the endoplasmic reticulum (ER), chloroplast, mitochondrion, endosome, and peroxisome are recruited for the formation of VRC-associated membranous structures. Previously, the 6,000-molecular-weight protein (6K) of plant potyviruses was shown to be an integral membrane protein that induces the formation of 6K-containing membranous vesicles at endoplasmic reticulum (ER) exit sites for potyvirus genome replication. Here, we present evidence that the 6K-induced vesicles predominantly target chloroplasts, where they amalgamate and induce chloroplast membrane invaginations. The vesicular transport pathway and actomyosin motility system are involved in the trafficking of the 6K vesicles from the ER to chloroplasts. Viral RNA, double-stranded RNA, and viral replicase components are concentrated at the 6K vesicles that associate with chloroplasts in infected cells, suggesting that these chloroplast-bound 6K vesicles are the site for potyvirus replication. Taken together, these results suggest that plant potyviruses sequentially recruit the ER and chloroplasts for their genome replication.The replication of eukaryotic positive-strand RNA viruses in infected cells is closely associated with unique virus-induced intracellular membranous vesicles (22). These membranous vesicles have been proposed to provide a scaffold for anchoring the virus replication complex (VRC), confine the process of RNA replication to a specific safeguarded cytoplasmic location, and prevent the activation of certain host defense mechanisms that can be triggered by double-stranded RNA (dsRNA) intermediates during virus replication (33, 47). Depending on the type of virus, the virus-induced membranous vesicles are derived from various intracellular organelles in the host. Many plant and animal viruses remodel and utilize the endoplasmic reticulum (ER) in VRCs (1, 6, 17, 33, 34, 36, 38, 39, 46). Other cellular organelles such as endosomes, lysomes, chloroplasts, peroxisomes, and mitochondria have also been suggest to be the replication site for togaviruses, tymoviruses, and tombusviruses, respectively (25, 27, 31). Given that the ER appears to be the site where the host cell translation machinery is hijacked for the biosynthesis of the first set of viral proteins, the subcellular location of virus replication (either in the vicinity of the ER or elsewhere) and the mechanism of transport to locations other than the ER are poorly understood.Plant potyviruses, accounting for ∼30% of known plant viruses including many agriculturally important viruses, e.g., Turnip mosaic virus (TuMV), Maize dwarf mosaic virus (MDMV), Tobacco etch virus (TEV), and Potato virus Y (PVY), are related to picornaviruses and picorna-like viruses (20, 21, 43). The potyviral genome is a single-stranded positive-sense RNA of about 10 kb in length and encodes at least 11 mature viral proteins (8, 43). Of these 11 proteins, the 6-kDa protein (designated 6K or 6K2) contains a central hydrophobic domain (35). In seminal work, Carrington and colleagues determined that 6K induces the formation of the ER-derived vesicles for TEV replication (35, 38). More recently, viral proteins required for replication and several host factors, namely, eukaryotic initiation factor (isoform) 4E, poly(A)-binding protein, eukaryotic elongation factor 1A, and heat shock cognate 70-3 protein, have been shown to associate with the TuMV 6K-induced vesicles (9, 41), raising the possibility that the potyviral 6K vesicles represent sites of viral genome replication. Furthermore, we have demonstrated that the biogenesis of the potyviral 6K vesicles occurs at COPII-accumulating ER exit sites (ERES) on the ER membrane (45). In this study, we further studied the trafficking of 6K-induced vesicles and found that the 6K-induced mobile vesicles trafficked predominantly from the ER to the periphery of chloroplasts. We show that these 6K vesicles docked on the outer chloroplast envelope and induced chloroplast invaginations. The chloroplast-associated 6K vesicles contained viral replicase components and dsRNA and were concentrated with viral RNA. We provide evidence that the early secretory pathway and actomyosin motility system were required for the trafficking of 6K vesicles from the ER to chloroplasts. These results suggest that plant potyviruses sequentially recruit the ER and chloroplasts for their genome replication. |
