Sequential Recruitment of the Endoplasmic Reticulum and Chloroplasts for Plant Potyvirus Replication

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 ϳ30% 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 protei...

Pumilio-Based RNA In Vivo Imaging

“…Pumilio-based RNA imaging has enabled subcellular detection of the mRNAs of mitochondrial NADH dehydrogenase and -actin, as well as retroviral RNA in mammalian cultured cells (20)(21)(22)(23), and of the genomes of Tobacco mosaic virus, Potato virus X and Turnip mosaic virus in live plant tissue (24)(25)(26)(27). Of particular relevance to the topic of this volume, Pumilio-based RNA imaging has recently shown that Potato virus X is present in membrane structures at the entrances of plasmodesmata that also contain the viral replicase (27).…”

“…This principle is the only form of Pumilio-based RNA imaging so far applied in plants ( [24][25][26][27].…”

“…An additional side-effect of RNA-independent formation of PUM-BiFC complexes is that these pre-formed complexes can be recruited to the target RNA by the interaction of only one of the two PUMHDs. Thus, RNAs can be imaged with only a single binding site (24,25) or when the orientation of PUMHD fusions would be expected to prevent proximity of split FP halves after RNA binding (see below) (22). Whilst this may sometimes be useful, it is not generally desirable, as it reduces the overall specificity of the PUM-BiFC system and eliminates the benefits of using two RNA binding domains.…”

“…PUM-BiFC imaging can be used in two different modes: PUMHDs can be modified to bind to the native RNA of interest (20,21,(23)(24)(25), or the RNA can be tagged with recognition motifs of previously characterised PUMHD variants (22,24,26,27,33) (Table 1). As described in the introduction, leaving the RNA unmodified has the advantage of avoiding potential RNA processing and localisation artefacts that could be caused by a tag, and by the expression of the tagged construct from an artificial genomic context.…”

“…Plant RNA virus genomes often contain native Pumilio binding sites that can be exploited for PUM-BiFC imaging (and may be targets of native Pumilio proteins involved in host defense responses) (24,25,42,43).…”