Nicotiana benthamiana plants were agroinoculated with an infectious cDNA clone of Turnip mosaic virus (TuMV) that was engineered to express a fluorescent protein (green fluorescent protein [GFP] or mCherry) fused to the viral 6K 2 protein known to induce vesicle formation. Cytoplasmic fluorescent discrete protein structures were observed in infected cells, corresponding to the vesicles containing the viral RNA replication complex. The vesicles were motile and aligned with microfilaments. Intracellular movement of the vesicles was inhibited when cells were infiltrated with latrunculin B, an inhibitor of microfilament polymerization. It was also observed that viral accumulation in the presence of this drug was reduced. These data indicate that microfilaments are used for vesicle movement and are necessary for virus production. Biogenesis of the vesicles was further investigated by infecting cells with two recombinant TuMV strains: one expressed 6K 2 GFP and the other expressed 6K 2 mCherry. Green-and red-only vesicles were observed within the same cell, suggesting that each vesicle originated from a single viral genome. There were also vesicles that exhibited sectors of green, red, or yellow fluorescence, an indication that fusion among individual vesicles is possible. Protoplasts derived from TuMV-infected N. benthamiana leaves were isolated. Using immunofluorescence staining and confocal microscopy, viral RNA synthesis sites were visualized as punctate structures distributed throughout the cytoplasm. The viral proteins VPg-Pro, RNA-dependent RNA polymerase, and cytoplasmic inclusion protein (helicase) and host translation factors were found to be associated with these structures. A single-genome origin and presence of protein synthetic machinery components suggest that translation of viral RNA is taking place within the vesicle.Positive-strand RNA viruses replicate their genomes on intracellular membranes. Extensive membrane rearrangements leading to cytoplasmic membranous structure production are observed during the infection cycle of many of these viruses (for a review, see reference 32). These virus-induced membrane structures vary greatly in origin, size, and shape. For instance, Flock House virus induces the formation of 50-nm vesicles (spherules), which are outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of approximately 10-nm diameter (24). On the other hand, poxviruses replicate in 1-to 2-m cytoplasmic foci known as DNA factories (43), which are bounded by rough endoplasmic reticulum (ER). These factories are not only the site of DNA synthesis but also of DNA transcription and RNA translation (21). Similarly, mimiviruses are huge doublestranded DNA viruses that replicate in giant cytoplasmic virus factories (45). Three-dimensional electron microscopic imaging has shown that coronavirus-induced membrane alterations define a reticulovesicular network of modified ER that integrates convoluted membranes, numerous interconnected double-membrane vesicle...

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...

The interaction between the viral protein linked to the genome (VPg) of turnip mosaic potyvirus (TuMV) and the translation eukaryotic initiation factor eIF(iso)4E of Arabidopsis thaliana has previously been reported. eIF(iso)4E binds the cap structure (m 7 GpppN, where N is any nucleotide) of mRNAs and has an important role in the regulation in the initiation of translation. In the present study, it was shown that not only did VPg bind eIF(iso)4E but it also interacted with the eIF4E isomer of A. thaliana as well as with eIF(iso)4E of Triticum aestivum (wheat). The interaction domain on VPg was mapped to a stretch of 35 amino acids, and substitution of an aspartic acid residue found within this region completely abolished the interaction. The cap analogue m 7 GTP, but not GTP, inhibited VPg-eIF(iso)4E complex formation, suggesting that VPg and cellular mRNAs compete for eIF(iso)4E binding. The biological significance of this interaction was investigated. Brassica perviridis plants were infected with a TuMV infectious cDNA (p35Tunos) and p35TuD77N, a mutant which contained the aspartic acid substitution in the VPg domain that abolished the interaction with eIF(iso)4E. After 20 days, plants bombarded with p35Tunos showed viral symptoms, while plants bombarded with p35TuD77N remained symptomless. These results suggest that VPg-eIF(iso)4E interaction is a critical element for virus production.

Unlike other positive-stranded RNA viruses that use either a 5 0 -cap structure or an internal ribosome entry site to direct translation of their messenger RNA, calicivirus translation is dependent on the presence of a protein covalently linked to the 5 0 end of the viral genome (VPg). We have shown a direct interaction of the calicivirus VPg with the cap-binding protein eIF4E. This interaction is required for calicivirus mRNA translation, as sequestration of eIF4E by 4E-BP1 inhibits translation. Functional analysis has shown that VPg does not interfere with the interaction between eIF4E and the cap structure or 4E-BP1, suggesting that VPg binds to eIF4E at a different site from both cap and 4E-BP1. This work lends support to the idea that calicivirus VPg acts as a novel 'cap substitute' during initiation of translation on virus mRNA. Keywords: calicivirus; eIF4E; translation; VPg EMBO reports (2005) 6, 968-972.

Schwann cells express low levels of myelin proteins in the absence of neurons. When Schwann cells and neurons are cultured together the production of myelin proteins is elevated, and myelin is formed. For peripheral myelin protein 22 (PMP22), the exact amount of protein produced is critical, because peripheral neuropathies result from its underexpression or overexpression. In this study we examined the effect of neurons on Schwann cell PMP22 production in culture and in peripheral nerve using metabolic labeling and pulse-chase studies as well as immunocytochemistry. Most of the newly synthesized PMP22 in Schwann cells is rapidly degraded in the endoplasmic reticulum. Only a small proportion of the total PMP22 acquires complex glycosylation and accumulates in the Golgi compartment. This material is translocated to the Schwann cell membrane in detectable amounts only when axonal contact and myelination occur. Myelination does not, however, alter the rapid turnover of PMP22 in Schwann cells. PMP22 may therefore be a unique myelin protein in that axonal contact promotes its insertion into the Schwann cell membrane and myelin without altering its rapid turnover rate within the cell.