The Vesicle-Forming 6K ₂ Protein of Turnip Mosaic Virus Interacts with the COPII Coatomer Sec24a for Viral Systemic Infection

Abstract

Positive-sense RNA viruses remodel host cell endomembranes to generate quasi-organelles known as "viral factories" to coordinate diverse viral processes, such as genome translation and replication. It is also becoming clear that enclosing viral RNA (vRNA) complexes within membranous structures is important for virus cell-to-cell spread throughout the host. In plant cells infected by turnip mosaic virus (TuMV), a member of the family Potyviridae, peripheral motile endoplasmic reticulum (ER)-derived viral vesicles are produced that carry the vRNA to plasmodesmata for delivery into adjacent noninfected cells. The viral protein 6K 2 is responsible for the formation of these vesicles, but how 6K 2 is involved in their biogenesis is unknown. We show here that 6K 2 is associated with cellular membranes. Deletion mapping and site-directed mutagenesis experiments defined a soluble N-terminal 12-amino-acid stretch, in particular a potyviral highly conserved tryptophan residue and two lysine residues that were important for vesicle formation. When the tryptophan residue was changed into an alanine in the viral polyprotein, virus replication still took place, albeit at a reduced level, but cell-to-cell movement of the virus was abolished. Yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitation experiments showed that 6K 2 interacted with Sec24a, a COPII coatomer component. Appropriately, TuMV systemic movement was delayed in an Arabidopsis thaliana mutant line defective in Sec24a. Intercellular movement of TuMV replication vesicles thus requires ER export of 6K 2 , which is mediated by the interaction of the N-terminal domain of the viral protein with Sec24a. IMPORTANCEMany plant viruses remodel the endoplasmic reticulum (ER) to generate vesicles that are associated with the virus replication complex. The viral protein 6K 2 of turnip mosaic virus (TuMV) is known to induce ER-derived vesicles that contain vRNA as well as viral and host proteins required for vRNA synthesis. These vesicles not only sustain vRNA synthesis, they are also involved in the intercellular trafficking of vRNA. In this investigation, we found that the N-terminal soluble domain of 6K 2 is required for ER export of the protein and for the formation of vesicles. ER export is not absolutely required for vRNA replication but is necessary for virus cell-to-cell movement. Furthermore, we found that 6K 2 physically interacts with the COPII coatomer Sec24a and that an Arabidopsis thaliana mutant line with a defective Sec24a shows a delay in the systemic infection by TuMV. P ositive-strand RNA viruses replicate on cellular membranes in order to achieve efficient virus production. These membranous structures have been termed "viral factories" and are associated with virus replication complexes (VRCs) (1, 2). Electron microscopy coupled to tomography has recently been used to get an elaborate three-dimensional view of animal virus-induced membranous structures. For instance, picorna-, corona-, and arteriviruses induce the formation of endopla...

Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection

“…6K1-GFP was found in both the cytoplasm and the nucleus (Fig. 5B, panel VI), consistent with the recent observation that the 6K1 of the potyvirus TuMV is a soluble protein when expressed ectopically (50).…”

“…5B, panels IV to VI). A similar observation was also reported for mCherry-tagged TuMV 6K1 in a recent study (50). However, in the presence of viral infection, 6K1 formed punctate structures (Fig.…”

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

“…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). Indeed, the systemic spread of TuMV is reduced in a Sec24a-defective Arabidopsis mutant (Jiang et al, 2015), and a dominant-negative Sar1 mutant (H74L) blocks the formation of 6K 2 vesicles and virus spread (Wei and Wang, 2008). Coxsackievirus B3 (CVB3), a picornavirus, also assembles its VRCs at ERES and a dominant-negative Sar1 mutant (T39N) inhibits CVB3 replication by ∼50% (Hsu et al, 2010).…”

“…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).…”

Recessive Resistance to Plant Viruses: Potential Resistance Genes Beyond Translation Initiation Factors

“…Since PVIP1 and PVIP2 interact with VPg proteins of other potyviruses, PVIPs-mediated resistance may also be effective against other potyviruses. Arabidopsis PCaP1 and COPII coatomer Sec24a interact with P3N-PIPO and 6K 2 of TuMV, respectively (Vijayapalani et al, 2012; Jiang et al, 2015). Both host factors are involved in distinct steps in TuMV cell-to-cell transport.…”