Subcellular Localisation, Protein Interactions, and RNA Binding of Potato mop-top virus Triple Gene Block Proteins

Abstract

Subcellular localisation, protein interactions, and RNA binding of the triple gene block proteins (TGBp) of Potato mop-top virus (PMTV) were studied. The 13-kDa (TGBp2) and 21-kDa (TGBp3) proteins with or without green fluorescent protein fused to their N-terminus, and the 51-kDa protein (TGBp1) were expressed individually from a recombinant Tobacco mosaic virus (TMV) vector. Fluorescent images and Western immunoblotting experiments of recombinant TMV-infected Nicotiana benthamiana cells suggested that TGBp2 and TGBp3 were associated with cellular endomembranes and that TGBp3 was associated with the cell wall, possibly located close to plasmodesmata. In Western blots, TGBp1 was detected in fractions containing the cell wall and those enriched for organelles and membranous structures. Self-interactions were demonstrated with all three proteins in yeast two-hybrid experiments, and a heterologous interaction was found between TGBp2 and TGBp3. No additional heterologous interactions were discovered between the different TGBp and none were detected in an in vitro binding assay. TGBp1 and TGBp2 but not TGBp3 were shown to bind ssRNA in a sequence nonspecific manner. The results support the model where TGBp2 and TGBp3 facilitate delivery and localisation of the ribonucleoprotein complex to the plasmodesmata. However, the process is facilitated by RNA-protein rather than protein:protein interactions between the TGBp1 in complex with viral RNA and membrane-localised TGBp2.

Two Plant–Viral Movement Proteins Traffic in the Endocytic Recycling Pathway

“…The N and C termini are predicted to be located in the cytoplasm, and there is a conserved hydrophilic motif (G-G-x-Y-R/K-D-G) in the intervening loop. The TGB2 of PMTV was shown previously to bind RNA in Northwestern gel blots (Cowan et al, 2002).…”

“…Transport along the recycling pathway depends on a functional actin cytoskeleton (Ayscough, 2004). TGB2 and TGB3 were shown previously to self-interact and also to interact with each other in the yeast two-hybrid system (Cowan et al, 2002). Therefore, it is possible that these interactions are important for homotypic vesicle fusion and multimerization of sorting signals that would improve the efficiency of internalization (Arneson and Miller, 1995).…”

“…Previously, we studied the TGB proteins of PMTV and described their subcellular localizations when expressed as N-terminal fusions to GFP from a Tobacco mosaic virus (TMV)-based vector (Cowan et al, 2002). In these experiments, both the PMTV GFP-TGB2 and GFP-TGB3 fusions were associated with cellular endomembranes, particularly the ER network and membranes surrounding the nucleus.…”

Análisis Funcional De Proteínas Codificadas Por El Virus De La Rotura Del Color De La Flor Del Pelargonium

“…En lo que respecta a las proteínas TGBp2 y TGBp3, son productos de menor tamaño y funcionan como proteínas integrales de membrana en ambas clases de virus [Tamai & Meshi, 2001;Cowan et al, 2002;Zamyatnin et al, 2002;Krishnamurthy et al, 2003;Gorshkova et al, 2003]. El producto TGBp2, cuyo tamaño oscila entre 11-14 kDa, posee dos secuencias hidrofóbicas internas que median su inserción en RE, dejando la región intermedia hidrofílica, muy conservada, dispuesta a modo de lazo hacia la cara luminal del orgánulo y los extremos N-t y C-t expuestos al citoplasma [Zamyatnin et al, 2006;Hsu et al, 2008;Lee et al, 2010].…”

“…En general., los procesos de dimerización parecen ser importantes para las asociaciones de muchas MP al RE; por ejemplo, se ha propuesto que dos subunidades de la MP del TMV (p30) pueden establecer interacciones entre las regiones ácidas y básicas de los extremos C-t extra-membranosos, permitiendo la formación de dímeros [Brill et al, 2004]. Asimismo, el direccionamiento a membranas de TGBp2 y TGBp3 está ligado a interacciones TGBp2:TGBp3 [Cowan et al, 2002;Samuels et al, 2007;Lim et al, 2008]. Entre los carmovirus, se ha apuntado que residuos de Cys situados en los extremos N-t de algunas DGBp2 podrían permitir la formación de puentes disulfuro, estabilizando uniones intermoleculares DGBp2:DGBp2 .…”