Membrane targeting sequences in tombusvirus infections

Infection of Nicotiana benthamiana cells with cymbidium ringspot (CymRSV) a nd carnation Italian ringspot (CIRV) viruses results in the formation of co nspicuous membranous bodies [multivesicular bodies (MVBs)], which develop f rom modified peroxisomes or mitochondria, respectively The organelle target ing signal is located in the proteins of 33 kDa (CymRSV) or 36 kDa (CIRV) e ncoded by ORF 1, which contain an N-terminal hydrophilic portion followed b y two predicted hydrophobic transmembrane segments. Biochemical analysis sh owed that the 33- and 36-kDa proteins are integral membrane proteins. By ex changing small portions of the ORF 1 sequence between the infectious full-l ength clones of the two viruses, hybrid constructs were obtained of which t he in vitro synthesized RNA was inoculated to N. benthamiana plants and pro toplasts. The structure of infectious clones suggested that both the N-term inal hydrophilic region and the transmembrane segments of the ORF 1-encoded proteins specify which organelle is involved in the synthesis of MVBs. Mut ational analysis of the CIRV 36-kDa protein also suggested the presence of an internal mitochondrial targeting sequence similar to that found in sever al normal host proteins that are synthesized in the cytoplasm and transport ed to mitochondria. The CymRSV 33-kDa protein did not contain the obvious c onsensus signals thought to be characteristic of proteins targeted to perox isomes, and an mitochondrial targeting sequence motif was not evident. (C) 1998 Academic Press.