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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...

Infection of Nicotiana benthamiana cells with cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) viruses results in the formation of conspicuous membranous bodies [multivesicular bodies (MVBs)], which develop from modified peroxisomes or mitochondria, respectively. The organelle targeting signal is located in the proteins of 33 kDa (CymRSV) or 36 kDa (CIRV) encoded by ORF 1, which contain an N-terminal hydrophilic portion followed by two predicted hydrophobic transmembrane segments. Biochemical analysis showed that the 33- and 36-kDa proteins are integral membrane proteins. By exchanging small portions of the ORF 1 sequence between the infectious full-length clones of the two viruses, hybrid constructs were obtained of which the in vitro synthesized RNA was inoculated to N. benthamiana plants and protoplasts. The structure of infectious clones suggested that both the N-terminal hydrophilic region and the transmembrane segments of the ORF 1-encoded proteins specify which organelle is involved in the synthesis of MVBs. Mutational analysis of the CIRV 36-kDa protein also suggested the presence of an internal mitochondrial targeting sequence similar to that found in several normal host proteins that are synthesized in the cytoplasm and transported to mitochondria. The CymRSV 33-kDa protein did not contain the obvious consensus signals thought to be characteristic of proteins targeted to peroxisomes, and an mitochondrial targeting sequence motif was not evident.

Open reading frame 1 in the viral genome of Cymbidium ringspot virus encodes a 33-kDa protein (p33), which was previously shown to localize to the peroxisomal membrane in infected and transgenic plant cells. To determine the sequence requirements for the organelle targeting and membrane insertion, the protein was expressed in the yeast Saccharomyces cerevisiae in native form (33K) or fused to the green fluorescent protein (33KGFP). Cell organelles were identified by immunolabeling of marker proteins. In addition, peroxisomes were identified by simultaneous expression of the red fluorescent protein DsRed containing a peroxisomal targeting signal and mitochondria by using the dye MitoTracker. Fluorescence microscopy showed the 33KGFP fusion protein concentrated in a few large bodies colocalizing with peroxisomes. These bodies were shown by electron microscopy to be composed by aggregates of peroxisomes, a few mitochondria and endoplasmic reticulum (ER) strands. In immunoelectron microscopy, antibodies to p33 labeled the peroxisomal clumps. Biochemical analysis suggested that p33 is anchored to the peroxisomal membrane through a segment of ca. 7 kDa, which corresponds to the sequence comprising two hydrophobic transmembrane domains and a hydrophilic interconnecting loop. Analysis of deletion mutants confirmed these domains as essential components of the p33 peroxisomal targeting signal, together with a cluster of three basic amino acids (KRR). In yeast mutants lacking peroxisomes p33 was detected in the ER. The possible involvement of the ER as an intermediate step for the integration of p33 into the peroxisomal membrane is discussed.