Turnip Mosaic Virus RNA Replication Complex Vesicles Are Mobile, Align with Microfilaments, and Are Each Derived from a Single Viral Genome

Abstract

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...

An unrecognized function for COPII components in recruiting the viral replication protein BMV 1a to the perinuclear ER

“…By contrast, the viral protein 6 kDa protein 2 (6K 2 ) of tobacco etch virus (TEV) and turnip mosaic virus (TuMV) is responsible for initiating the assembly of 6K 2 -induced vesicles (6K 2 -vesicles). These vesicles serve as VRCs, exit ER membranes and eventually assemble into large granule structures at chloroplasts in addition to playing a possible role in cell-to-cell movement (Cheng et al, 2015;Cotton et al, 2009;Jiang et al, 2015;Wei and Wang, 2008;Wei et al, 2010). Out of the three Arabidopsis Sec24 isoforms (Sec24a, Sec24b and Sec24c) (Marti et al, 2010), 6K 2 interacts with Sec24a to assemble 6K 2 -vesicles at ER exit sites (ERES).…”

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

“…PCR involved in the gateway cloning was carried out with Phusion DNA polymerase (NEB, Pickering, Ontario, Canada). The recombinant TuMV infectious clone tagged with a chimeric gene containing the 6K-coding sequence fused in frame with the green fluorescent protein (GFP)-coding sequence (TuMV::6K-GFP) was described previously (9,41). The 6K, NIb, and cylindrical inclusion (CI) cistrons of TuMV were amplified from infectious clone TuMV::6K-GFP by PCR.…”

“…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).…”

ArabidopsisPumilio protein APUM5 suppressesCucumber mosaic virusinfection via direct binding of viral RNAs

“…On the other hand, Turnip mosaic virus (TuMV) is a single-stranded Arabidopsis-infecting RNA virus that does have a poly(A) tail in its 3′ UTR. TuMV also contains putative Pumilio-binding core motifs in its genome and 3′ UTR (16). In TuMV-UK1 3′ UTR, putative "UGUA" core sequences were found (Fig.…”