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Journal of Virology, October 2002, p. 10485-10496, Vol. 76, No. 20
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.20.10485-10496.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Frédérique Weber-Lotfi,1 André Dietrich,1* Marcello Russo,2 and Luisa Rubino2

Institut de Biologie Moléculaire des Plantes du CNRS, Université Louis Pasteur, 67084 Strasbourg, France,1 Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, and Centro di Studio del CNR sui Virus e le Virosi delle Colture Mediterranee, 70126 Bari, Italy2

Received 18 April 2002/ Accepted 17 June 2002

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.

* Corresponding author. Mailing address: Institut de Biologie Moléculaire des Plantes du CNRS, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France. Phone: 33 3 88 41 72 41. Fax: 33 3 88 61 44 42. E-mail: andre.dietrich{at}ibmp-ulp.u-strasbg.fr.

Journal of Virology, October 2002, p. 10485-10496, Vol. 76, No. 20
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.20.10485-10496.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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