Mutations in the Central Domain of Potato Virus X TGBp2 Eliminate Granular Vesicles and Virus Cell-to-Cell Trafficking

Abstract

Most RNA viruses remodel the endomembrane network to promote virus replication, maturation, or egress. Rearrangement of cellular membranes is a crucial component of viral pathogenesis. The PVX TGBp2 protein induces vesicles of the granular type to bud from the endoplasmic reticulum network. Green fluorescent protein (GFP) was fused to the PVX TGBp2 coding sequence and inserted into the viral genome and into pRTL2 plasmids to study protein subcellular targeting in the presence and absence of virus infection. Mutations were introduced into the central domain of TGBp2, which contains a stretch of conserved amino acids. Deletion of a 10-amino-acid segment (m2 mutation) overlapping the segment of conserved residues eliminated the granular vesicle and inhibited virus movement. GFP-TGBp2m2 proteins accumulated in enlarged vesicles. Substitution of individual conserved residues in the same region similarly inhibited virus movement and caused the mutant GFP-TGBp2 fusion proteins to accumulate in enlarged vesicles. These results identify a novel element in the PVX TGBp2 protein which determines vesicle morphology. In addition, the data indicate that vesicles of the granular type induced by TGBp2 are necessary for PVX plasmodesmata transport.Most positive-strand RNA viruses cause specific changes in membrane architecture, creating distinct compartments for virus replication complexes. The types of membrane modifications include proliferations, invaginations, and novel vesicles or spherules. Proliferations are seen under the electron microscope as an expansion or increase in membrane layers. Invaginations, vesicles, or spherules often derive from the endoplasmic reticulum (ER), nuclear envelope, or organelles. These virus-induced compartments protect the replicating virus from host proteases and other defenses.Poliovirus (PV), vaccinia virus, tobacco etch virus, cowpea mosaic virus, and brome mosaic virus are also examples of viruses which cause proliferation and invaginations of the ER for replication (8,9,12,20,41,45,48,54). The membrane invaginations are containers for the viral replicase protecting the replication complexes from cellular degrading enzymes. Brome mosaic virus 1a is responsible for induction of vesicles, while the nature of the structures formed vary with the ratio of 1a:2a proteins (50, 51). When 2a is expressed at low levels, vesicles are induced. However, with increasing 2a concentrations, membranes accumulate in stacks rather than vesicles (51).The tobacco mosaic virus (TMV) 126-kDa replicase protein induces membraneous bodies to form in the absence of other TMV proteins. The TMV movement protein associates with these bodies and transports them along the microfilament network toward the periphery of the cell and, possibly, across the plasmodesmata (25,30). Thus, TMV is the first plant virus that has been reported to involve membrane-bound replication complexes in the cell-to-cell transport pathway.Beyond TMV, many plant viruses involve the endomembrane system in virus intracellular and intercellular m...

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

“…Vesicular structures contained ribosomes, were immunoactive from GFP and BiP antigens (chaperon protein present in ER). Deletion of conservative amino acids in TGBp2 central region (located between 2 transmembrane domains) blocked the accumulation of GFP-TGBp2 in small, granular vesicles and inhibited cell-to-cell movement of the virus (Ju et al 2007). Such data indicate that vesicular structures induced by TGBp2 PVX are necessary for virus movement (Ju et al 2007).…”

“…Deletion of conservative amino acids in TGBp2 central region (located between 2 transmembrane domains) blocked the accumulation of GFP-TGBp2 in small, granular vesicles and inhibited cell-to-cell movement of the virus (Ju et al 2007). Such data indicate that vesicular structures induced by TGBp2 PVX are necessary for virus movement (Ju et al 2007). In tobacco plants, where the construct GFP-TGBp3 was subject to expression, fluorescence was connected .…”

“…Virions or viral RNA can be transported to PD during later stages of infection inside the cell. If TGBp2 and TGBp3 coordinate the transport of TGBp1-CP-vRNA complex along ER into PD (Morozov and Solovyev 2003;Lucas 2006), available data illustrating that vesicles inducing TGBp2 PVX are vital for cell-to-cell transfer (Ju et al 2007) bring to mind a question about the contents of these vesicles. Do they transfer infection agents from cell to cell?…”

Análisis funcional y localización subcelular de las proteínas implicadas en el movimiento intra e intercelular del virus de las manchas necróticas del melón (MNSV)

“…On the other hand, in two groups of positive-strand RNA viruses (potex-like and hordeilike viruses), the movement function is provided by the proteins encoded by the so-called triple gene block (TGB) including an RNA binding protein with helicase activity (TGBp1) and two putative small membrane-associated proteins (TGBp2 and TGBp3). All three proteins move from ER to the cellular periphery generating ER-derived vesicles which also travel along actin/myosin microfilaments and it is also known that later, the membrane-associated MPs can be recycled making use of the endocityc pathway (Haupt et al, 2005;Ju et al, 2007;.…”

“…On the other hand, in two groups of positive-strand RNA viruses (potex-like and hordeilike viruses), the movement function is provided by the proteins encoded by the so-called triple gene block (TGB) including an RNA binding protein with helicase activity (TGBp1) and two putative small membrane-associated proteins (TGBp2 and TGBp3). All three proteins move from ER to the cellular periphery generating ER-derived vesicles which also travel along actin/myosin microfilaments and it is also known that later, the membrane-associated MPs can be recycled making use of the endocityc pathway (Haupt et al, 2005;Ju et al, 2007;.With the purpose of going further in the knowledge of the different routes plant viruses hijack to reach the PDs we use one of the most simple multi-component transport system ______________________________________________________________________________________C Ca ap pí ít tu ul lo o I IV V 157 identified to date such as those of the Carmovirus genus, which is extremely reduced in the case of Melon necrotic spot virus, MNSV (Hibi and Furuki, 1985). The MNSV has a singlestranded RNA genome of 4.3 kb long which code for five functionally characterized proteins:…”