RNA-binding properties and membrane insertion of Melon necrotic spot virus (MNSV) double gene block movement proteins

Navarro, J. A.; Genovés, Ainhoa; Climent, Joan; Saurı́, Ana; Martínez-Gil, Luis; Mingarro, Ismael; Pallás, Vicente

doi:10.1016/j.virol.2006.07.040

Cited by 39 publications

(89 citation statements)

References 60 publications

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“…3A, untreated lanes) upon centrifugation of the microsome-containing translation reaction mixture, indicating that it could be either a membraneassociated (peripheral or integral) protein or a lumenally translocated protein. To differentiate between these possibilities, the translation reaction mixtures were first washed with sodium carbonate (pH 11.5), which renders microsomes into membranous sheets, releasing the soluble luminal proteins (25,29). As shown in Fig.…”

Section: Sequence Analysis Of the Pnrsv Mpmentioning

confidence: 99%

Plant Virus Cell-to-Cell Movement Is Not Dependent on the Transmembrane Disposition of Its Movement Protein

Martínez-Gil

Sánchez‐Navarro

Cruz

et al. 2009

J Virol

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The cell-to-cell transport of plant viruses depends on one or more virus-encoded movement proteins (MPs). Some MPs are integral membrane proteins that interact with the membrane of the endoplasmic reticulum, but a detailed understanding of the interaction between MPs and biological membranes has been lacking. The cell-to-cell movement of the Prunus necrotic ringspot virus (PNRSV) is facilitated by a single MP of the 30K superfamily. Here, using a myriad of biochemical and biophysical approaches, we show that the PNRSV MP contains only one hydrophobic region (HR) that interacts with the membrane interface, as opposed to being a transmembrane protein. We also show that a proline residue located in the middle of the HR constrains the structural conformation of this region at the membrane interface, and its replacement precludes virus movement.Plant viruses encode movement proteins (MPs) that mediate the intra-and intercellular spread of the viral genome via plasmodesmata, membranous channels that traverse the walls of plant cells and enable intercellular transport and communication. There is a range of diversity in the number and type of viral proteins required for viral movement (21). Research on tobacco mosaic virus (TMV) has played a leading role in understanding MP activity (2). The genome of TMV encodes a single 30-kDa multidomain protein, the namesake of the 30K superfamily (7). Viral RNA is associated with the membrane of the endoplasmic reticulum (ER) and microtubules in the presence of this MP (23,30).A large number of plant viruses have 30K MPs, which share common abilities, including binding nucleic acids, localizing and increasing the size exclusion limit of plasmodesmata, and interacting with the ER membrane. A topological model has been proposed in which the TMV MP has two putative transmembrane (TM) helices, both the N and C termini oriented toward the cytoplasm, and a short loop exposed in the ER lumen (4). There is less experimental information for other 30K MPs, but they are likely to have some membrane interaction.Direct experimental evidence of the integration of MPs into the membrane has been obtained only for small hydrophobic MPs that do not belong to the 30K superfamily. There are two TM segments in the p9 protein of carnation mottle virus (41), whereas the p6 protein of beet yellow virus (29) and the p7B protein of melon necrotic spot virus (22) have a single TM segment. In viruses with genomes that include three partially overlapping open reading frames, termed the triple-gene block (TGB), all three TGB proteins are required for movement where the two smaller proteins, TGBp2 and TGBp3, are also TM proteins (24). Furthermore, cross-linking experiments with carnation mottle virus p9 protein demonstrated that its membrane insertion occurs cotranslationally in a signal recognition particle-dependent manner and throughout the cellular membrane integration components, the translocon (33, 34).Prunus necrotic ringspot virus (PNRSV) is a tripartite, positive-strand RNA virus in the genus Ilarvirus ...

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Section: Sequence Analysis Of the Pnrsv Mpmentioning

confidence: 99%

Plant Virus Cell-to-Cell Movement Is Not Dependent on the Transmembrane Disposition of Its Movement Protein

Martínez-Gil

Sánchez‐Navarro

Cruz

et al. 2009

J Virol

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“…11,46 The K42D, K46D, and D74K site-directed mutagenesis were done using the QuikChange mutagenesis kit from Stratagene following the manufacturer's protocol. All DNA manipulations were confirmed by sequencing of plasmid DNAs.…”

Section: Dna Manipulationsmentioning

confidence: 99%

Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion

Bañó‐Polo

Martínez-Gil

Wallner

et al. 2013

Journal of Molecular Biology

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α-Helical hairpins, consisting of a pair of closely spaced transmembrane (TM) helices that are connected by a short interfacial turn, are the simplest structural motifs found in multi-spanning membrane proteins. In naturally occurring hairpins, the presence of polar residues is common and predicted to complicate membrane insertion. We postulate that the pre-packing process offsets any energetic cost of allocating polar and charged residues within the hydrophobic environment of biological membranes. Consistent with this idea, we provide here experimental evidence demonstrating that helical hairpin insertion into biological membranes can be driven by electrostatic interactions between closely separated, poorly hydrophobic sequences. Additionally, we observe that the integral hairpin can be stabilized by a short loop heavily populated by turn-promoting residues. We conclude that the combined effect of TM-TM electrostatic interactions and tight turns plays an important role in generating the functional architecture of membrane proteins and propose that helical hairpin motifs can be acquired within the context of the Sec61 translocon at the early stages of membrane protein biosynthesis. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains from primary structures.

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“…En el caso de las DGBp1, análisis comparativos de secuencia han definido la existencia de tres dominios estructurales: (1) uno N-t apenas estructurado, que se encuentra muy poco conservado entre proteínas homólogas, (2) otro C-t, plegado a modo de lámina-β y altamente conservado en proteínas equivalentes y (3) una región central α-helicoidal Akgoz et al, 2001;Vilar et al, 2001Vilar et al, , 2005Navarro et al, 2006]. En lo que respecta a las DGBp2, uno de los aspectos más destacables de su estructura es la presencia de una o dos regiones con un alto porcentaje de aminoácidos hidrofóbicos que pueden comportarse como dominios TM; además, en todos los casos se predicen plegamientos en forma de lámina-β en el extremo C-t [Vilar et al, 2001;Navarro et al, 2006]. A continuación se describirán algunas características funcionales de estas proteínas y, más brevemente, de otras de la familia, prestando especial atención a las propiedades de unión a RNA y a la localización subcelular de las mismas.…”

Section: Proteínas Implicadas En El Movimiento En La Familia Tombusviunclassified

“…Propiedades similares de unión a ssRNAs, y en menor medida a otros ácidos nucleicos, fueron descritas posteriormente para la proteínas p7 del CarMV y p7A del MNSV Navarro et al, 2006]. En los dos últimos casos se ha identificado la región responsable de esta característica, que corresponde al dominio central rico en aminoácidos básicos y que adopta un plegamiento en hélice-α, aunque en la proteína p7A del MNSV no se descarta la implicación de motivos adicionales situados fuera de este dominio [Vilar et al, 2001[Vilar et al, , 2005Navarro et al, 2006]. Los datos obtenidos sugieren que los residuos cargados positivamente participan en la interacción directa con el ácido nucleico y que otros hidrofóbicos funcionan estabilizando los complejos RNA-proteína.…”

Section: Propiedades De Unión a Rnaunclassified

“…Los datos obtenidos sugieren que los residuos cargados positivamente participan en la interacción directa con el ácido nucleico y que otros hidrofóbicos funcionan estabilizando los complejos RNA-proteína. Las constantes de disociación (Kd) estimadas para los complejos p7(CarMV)-ssRNA y p7A(MNSV)-ssRNA se aproximan a las de otras MPs y están dentro del orden μM Vilar et al, 2001;Navarro et al, 2006]. El hallazgo de propiedades de unión a ácidos nucleicos en algunas DGBp1 y de interacción con membranas en las DGBp2, llevó a proponer un modelo de reparto de funciones entre estas proteínas durante el movimiento viral.…”

Section: Propiedades De Unión a Rnaunclassified

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

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RNA-binding properties and membrane insertion of Melon necrotic spot virus (MNSV) double gene block movement proteins

Cited by 39 publications

References 60 publications

Plant Virus Cell-to-Cell Movement Is Not Dependent on the Transmembrane Disposition of Its Movement Protein

Plant Virus Cell-to-Cell Movement Is Not Dependent on the Transmembrane Disposition of Its Movement Protein

Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion

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

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