The Potato Virus X TGBp2 Movement Protein Associates with Endoplasmic Reticulum-Derived Vesicles during Virus Infection

Abstract

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virusinfected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.Plant viruses use the cellular endomembrane system to support virus replication (Schaad et al., 1997;Reichel and Beachy, 1998;Carette et al., 2002aCarette et al., , 2002bSchwartz et al., 2002). Among positive-strand RNA viruses, the viral replicase is typically anchored to membranes associated with the endoplasmic reticulum (ER), the endocytic pathway, or cellular organelles (Rubino and Russo, 1998;Mas and Beachy, 1999;Rubino et al., 2001;Schwartz et al., 2002Schwartz et al., , 2004. Many viral replicationassociated proteins cause reorganization of cellular membranes with the purpose of creating centers that protect the viral replication complexes (VRCs) from cellular nucleases and host defenses (Schwartz et al., 2002(Schwartz et al., , 2004Ding et al., 2004). Specifically, the replication complexes of plant viruses belonging to the genera Potyvirus, Comovirus, Dianthovirus, Pecluvirus, and Bromovirus cause proliferation and vesiculation of either the perinuclear or the cortical ER (Schaad et al., 1997;Dunoyer et al., 2002;Lee and Ahlquist, 2003). The replicases of cowpea mosaic virus and red clover necrotic mosaic virus induce ER proliferation (Carette et al., 2002a(Carette et al., , 2002bTurner et al., 2004). The best-studied example is the brome mosaic virus (BMV) 1a replicase, which localizes to the ER and induces membrane i...

Mutation of a chloroplast-targeting signal in Alternanthera mosaic virus TGB3 impairs cell-to-cell movement and eliminates long-distance virus movement

“…Subcellular localization of several class I (hordeivirus-like) and class II (potexvirus-like) TGB proteins has been investigated by using fluorescent gene fusions (Haupt et al, 2005;Ju et al, 2005;Lim et al, 2009;Lough et al, 1998;Solovyev et al, 2000). To assess localization of AltMV TGB3, we expressed TGB3 from pGDR and pGDG (Goodin et al, 2002) or pHVLR and pHVLG vectors for Agrobacteriummediated transient expression of DsRed or green fluorescent protein (GFP) fusions (Fig.…”

“…Several reports have addressed the contributions of TGB proteins to movement for WClMV (Lough et al, 1998), PVX (Ju et al, 2005;Krishnamurthy et al, 2003;Lough et al, 2000;Solovyev et al, 2000;Tamai & Meshi, 2001) and BaMV (Lin et al, 2006), and describe trans complementation of potexviral genomes with defects within the TGB through delivery of functional TGB constructs by using particle bombardment (Morozov et al, 1997), transgenic plants (Lough et al, 1998) or satellite RNA (Lin et al, 2006). Despite this body of work, 'a full understanding of the specific requirement of TGB3 and the functional interchangeability of TGB3 among different potexviruses for intercellular movement remains elusive' (Lin et al, 2006).…”

Cell-to-cell movement of three genera (+) ss RNA plant viruses

“…Mutation disrupting membrane associations of these proteins also inhibit virus movement, indicating that ER association is important . GFP to TGBp3 fusion and introducing a construct into a PVX genome in protoplasts of inoculated plants resulted in constructs fluorescence mainly in small granular vesicles and in ER (Ju et al 2005). Granular vesicles accumulate on actin filaments, which suggest that they may move along the cytoskeleton to PD.…”

“…Model in which vesicles transport viral RNA to PD seems to be rather in opposition to the theory on the movement of TGBp1-CP-vRNA complex along ER to PD. It is also possible that TGBp2-induced vesicles play a role in modulating the ER stress responses (Ju et al 2005) or other events in the virus life cycle, thereby enabling virus cell-to-cell movement.…”

“…Potexvirus code two proteins associated with ER: TGBp2 and TGBp3 Ju et al 2005). They are the core of the movement process and so far no direct connection with replication process has been found.…”