Biogenesis of Cytoplasmic Membranous Vesicles for Plant Potyvirus Replication Occurs at Endoplasmic Reticulum Exit Sites in a COPI- and COPII-Dependent Manner

Abstract

Single-stranded positive-sense RNA viruses induce the biogenesis of cytoplasmic membranous vesicles, where viral replication takes place. However, the mechanism underlying this characteristic vesicular proliferation remains poorly understood. Previously, a 6-kDa potyvirus membrane protein (6K) was shown to interact with the endoplasmic reticulum (ER) and to induce the formation of the membranous vesicles. In this study, the involvement of the early secretory pathway in the formation of the 6K-induced vesicles was investigated in planta. By means of live-cell imaging, it was found that the 6K protein was predominantly colocalized with Sar1, Sec23, and Sec24, which are known markers of ER exit sites (ERES). The localization of 6K at ERES was prevented by the coexpression of a dominant-negative mutant of Sar1 that disables the COPII activity or by the coexpression of a mutant of Arf1 that disrupts the COPI complex. The secretion of a soluble secretory marker targeting the apoplast was arrested at the level of the ER in cells overexpressing 6K or infected by a potyvirus. This blockage of protein trafficking out of the ER by 6K and the distribution of 6K toward the ERES may account for the aggregation of the 6K-bound vesicles. Finally, virus infection was reduced when the accumulation of 6K at ERES was inhibited by impairing either the COPI or COPII complex. Taken together, these results imply that the cellular COPI and COPII coating machineries are involved in the biogenesis of the potyvirus 6K vesicles at the ERES for viral-genome replication.

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).…”

Subcellular localization and membrane association of the replicase protein of grapevine rupestris stem pitting-associated virus, family Betaflexiviridae

“…This can be through: (i) a viral protein possessing a transmembrane domain, as in the case of the 6 kDa protein of potyviruses (Schaad et al, 1997;Wei & Wang, 2008;Wei et al, 2010); (ii) interaction with an integral membrane protein from the host, as in the case of TMV, which requires the transmembrane proteins TOM1 and TOM3 (Yamanaka et al, 2000(Yamanaka et al, , 2002; and (iii) monotopic membrane association by a virus-encoded protein containing an IPM anchor (an amphipathic a-helix), as in the case of SFV (Salonen et al, 2005;Spuul et al, 2007) and BMV (Liu et al, 2009). An amphipathic a-helix contains a hydrophobic face that inserts into one leaflet of the lipid bilayer, thus achieving integral membrane association (Sapay et al, 2006;Spuul et al, 2007).…”

Detection, Understanding and Controlof Soybean Mosaic Virus

“…Subsequently, uncoating of the viral RNAs in the cytoplasm, translation of the polyprotein and replication of the viral genome occur. Accumulated evidence suggests the replication of eukaryotic positive-strand RNA viruses including plant potyviruses is associated with intracellular membranous structures (Wei and Wang, 2008). 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 to prevent the activation of host defense responses, and to recruit the components required for replication and maintain the proper concentrations of these components (Wileman, 2006).…”

“…It has been shown that in potyvirus-infected plant cells, these vesicular compartments contain a number of host proteins, i.e., heat shock cognate 70-3 (Hsc70-3), poly(A)-binding protein (PABP), eEF1A and eIF(iso)4E, and several non-structural viral proteins, such as the 6K2, NIb (the viral RNA dependent RNA polymerase, RdRp), NIa (including NIa-VPg or NIa-Pro or as a precursor protein) and CI Thivierge et al, 2008;Cotton et al, 2009;Wei et al, 2010a). The 6K2 (also called 6K) protein is an integral membrane protein that induces the formation of 6K-containing membranous vesicles at ERES (Wei and Wang, 2008). These ER-derived vesicles further target chloroplasts where they amalgamate and induce chloroplast membrane invaginations (Wei and Wang, 2008;Wei et al, 2010a).…”

“…The 6K2 (also called 6K) protein is an integral membrane protein that induces the formation of 6K-containing membranous vesicles at ERES (Wei and Wang, 2008). These ER-derived vesicles further target chloroplasts where they amalgamate and induce chloroplast membrane invaginations (Wei and Wang, 2008;Wei et al, 2010a). Thus, 6K plays a pivotal role in the formation and targeting of VRC-associated vesicles.…”