Mitochondrial Targeting and Membrane Anchoring of a Viral Replicase in Plant and Yeast Cells

Abstract

Replication of the Carnation Italian ringspot virus genomic RNA in plant cells occurs in multivesicular bodies which develop from the mitochondrial outer membrane during infection. ORF1 in the viral genome encodes a 36-kDa protein, while ORF2 codes for the 95-kDa replicase by readthrough of the ORF1 stop codon. We have shown previously that the N-terminal part of ORF1 contains the information leading to vesiculation of mitochondria and that the 36-kDa protein localizes to mitochondria. Using infection, in vivo expression of green fluorescent protein fusions in plant and yeast cells, and in vitro mitochondrial integration assays, we demonstrate here that both the 36-kDa protein and the complete replicase are targeted to mitochondria and anchor to the outer membrane with the N terminus and C terminus on the cytosolic side. Analysis of deletion mutants indicated that the anchor sequence is likely to correspond approximately to amino acids 84 to 196, containing two transmembrane domains. No evidence for a matrix-targeting presequence was found, and the data suggest that membrane insertion of the viral proteins is mediated by an import receptor-independent signal-anchor mechanism relying on the two transmembrane segments and multiple recognition signals present in the N-terminal part of ORF1.The genomes of positive-stranded RNA viruses from a number of supergroups are replicated in association with intracellular membranes (3). Depending on the virus, a variety of membrane systems can be concerned in infected cells, including the plasma membrane, endoplasmic reticulum, vacuole, chloroplasts, mitochondria, peroxisomes, and lysosomes (19,22,32,38,42,47). Little is known about the molecular mechanisms supporting these virus-cell interactions, which are critical for the development of infection. Membrane interaction with both viral proteins and host-encoded factors (50) has been proposed. Viral RNA replication is often associated with infection-specific membrane proliferation and/or vesiculation.Plant infection with Carnation Italian ringspot virus (CIRV), a member of the genus Tombusvirus in the family Tombusviridae, triggers the development of conspicuous membrane bodies from modified mitochondria (11). The CIRV genome is composed of a monopartite, positive-sense RNA genome of 4,760 nucleotides (GenBank accession number X85215). It is not polyadenylated, lacks a 5Ј cap structure, and contains five functional open reading frames (ORFs) (36) (Fig. 1). The 5Ј-most ORF (ORF1) in the CIRV genome encodes a 36-kDa protein (36K protein), while ORF2 codes for a 95-kDa protein (95K), which is expressed by readthrough of the amber stop codon of ORF1. Readthrough occurs at a frequency of about 10% in plant cells. Both pre-and complete readthrough products are essential for viral replication and were shown to contain the eight conserved motifs (PI to PVIII) of RNAdependent RNA polymerases of supergroup II of the positivestranded RNA viruses (16,40). ORF3 encodes the coat protein of 41 kDa, and the two nested ORFs 4 and 5 code for 21...

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

“…Para algunos virus de plantas de ssRNA(+), tanto la RdRp como una proteína auxiliar de la replicación conservan por separado la capacidad de dirigirse a los orgánulos intracelulares; sin embargo, en otros casos es necesaria la participación simultánea de ambas proteínas. Una tercera posibilidad, es que la proteína auxiliar de la replicación sea responsable, en última instancia, del direccionamiento de la RdRp, del vRNA y de todo el complejo replicativo hacia los sitios de replicación [Restrepo-Hartwig & Ahlquist, 1999;Chen & Ahlquist, 2000;Weber-Lotfi et al, 2002;Prod´homme et al, 2003;Jakubiec et al, 2004;Nagy & Pogany, 2008]. Excepcionalmente, el anclaje del complejo replicativo puede no estar mediado directamente por las replicasas virales, sino por una proteína del huésped [Hagiwara et al, 2003;Yamanaka et al, 2000].…”

“…La información disponible actualmente acerca de las replicasas de los miembros de la familia Tombusviridae se ha obtenido fundamentalmente a partir de los trabajos realizados con varios miembros del género Tombusvirus [Bleve-Zacheo et al, 1997;Oster et al, 1998;Rubino et al, 2001;Weber-Lotfi et al, 2002;Panaviene et al, 2003;Navarro et al, 2004;Panavas et al, 2005a;] y con una especie del género Dianthovirus [Bates et al, 1995;Turner et al, 2004;Takeda et al, 2005;Okamoto et al, 2008;Mine et al, 2010b]. Estas investigaciones se han centrado en los requerimientos de secuencias y las actividades enzimáticas de la RdRp, en las propiedades de unión a ácidos nucleicos de ambas replicasas, su localización subcelular, la búsqueda de factores del huésped implicados en la replicación y en la interacción entre ambas proteínas.…”

“…De hecho, en distintos casos se ha constatado la co-localización de las replicasas en los sitios donde tiene lugar la síntesis del vRNA. En el género Tombusvirus, las proteínas p36 y p95 del CIRV co-localizan en mitocondrias [Weber-Lotfi et al, 2002;Pantaleo et al, 2004] y sus homólogas, las proteínas p33 y p92 del CymRSV [Lupo et al, 1994], del TBSV [McCartney et al, 2005] y del CNV [Panavas et al, 2005a], lo hacen en peroxisomas. Igualmente, en el género Dianthovirus se ha observado que ambas replicasas del RCNMV, p27 y p88, co-localizan en el RE [Turner et al, 2004].…”

“…In many cases, α-helices that function as transmembrane domains (TMs) play a critical role in both targeting and integration to specific membranes. That is the case of CIRV p36, that has been proposed to associate to the mitochondrial outer membrane through two TMs and multiple recognition signals present at the N-terminus that might function cooperatively as a so-called signal loop-anchor type mitochondrial targeting sequence [Weber-Lotfi et al, 2002;Hwang et al, 2008].…”

“…Thus, the ORF1 products of Red clover necrotic mosaic virus (RCNMV, genus Dianthovirus) and of Panicum mosaic virus (PMV, genus Panicovirus) associate to membranes of the endoplasmic reticulum [Batten et al, 2006b;Turner et al, 2004], whereas the ORF1 products of TBSV, Cymbidium ringspot virus (CymRSV) and Cucumber necrosis virus (CNV) in the genus Tombusvirus, are targeted to peroxisomes [McCartney et al, 2005;Navarro et al, 2004;Panavas et al, 2005]. Despite Carnation Italian ringspot virus (CIRV) belongs also to genus Tombusvirus, its ORF1 product (p36) is sorted to mitochondria [Weber-Lotfi et al, 2002]. Most of these proteins induce organelle aggregation and/or proliferation of the membranes they associate with and seem to be truly integrated in the corresponding membranes.…”