Positive-strand RNA virus replication always occurs in association with rearranged host cell membranes. In infected plants, replication of tombusviruses takes place in membranous structures, known as multivesicular bodies (MVBs) which originate from vesiculation of the limiting membrane of peroxisomes or of the mitochondrial outer membrane. We investigated the mechanism of vesicle formation on mitochondria in Carnation Italian ringspot virus (CIRV) infections. The genome of CIRV consists of a 4.8 kb single-stranded RNA molecule encapsidated in icosahedral particles. The genome lacks a 5' cap structure, is not polyadenylated at the 3' end and contains five open reading frames (ORFs). ORF1- and ORF2-encoded p36 and p95 proteins are essential for viral replication. In particular, p95 contains the conserved motifs of RNA-dependent RNA polymerases and p36 the motifs for viral RNA binding and recruitment to replication sites. ORF 3 codes for the p41 coat protein, ORFs 4 and 5 encode p22, required for cell-to-cell movement of the virus in infected plants, and p19, which is a suppressor of virus induced gene silencing, respectively. The requirements for the formation of MVBs were studied in cells infected by cis replicating wild type or defective CIRV genomes, or expressing the p36 and p95 replicase proteins from a non replicatable genome but able to support in trans the replication of defective interfering RNAs. It was ascertained that MVB developed in cells transfected with CIRV defective genomes that, although expressing only p36 and p95, were able to replicate.