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Journal of Virology, February 2001, p. 1879-1887, Vol. 75, No. 4
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.4.1879-1887.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Alfalfa Mosaic Virus Replicase Proteins P1 and P2 Interact and Colocalize at the Vacuolar Membrane

Maurice W. Van Der Heijden,1 Jan E. Carette,2 Poula J. Reinhoud,1 Anita Haegi,1 and John F. Bol1,*

Institute of Molecular Plant Sciences, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden,1 and Laboratory of Molecular Biology, Wageningen University & Research Centre, 6703 HA Wageningen,2 The Netherlands

Received 14 August 2000/Accepted 9 November 2000

Replication of Alfalfa mosaic virus (AMV) RNAs depends on the virus-encoded proteins P1 and P2. P1 contains methyltransferase- and helicase-like domains, and P2 contains a polymerase-like domain. Coimmunoprecipitation experiments revealed an interaction between in vitro translated-P1 and P2 and showed that these proteins are present together in fractions with RNA-dependent RNA polymerase activity. A deletion analysis in the yeast two-hybrid system showed that in P1 the C-terminal sequence of 509 amino acids with the helicase domain was necessary for the interaction. In P2, the sequence of the N-terminal 241 aa was required for the interaction. In infected protoplasts, P1 and P2 colocalized at a membrane structure that was identified as the tonoplast (i.e., the membrane that surrounds the vacuoles) by using a tonoplast intrinsic protein as a marker in immunofluorescence studies. While P1 was exclusively localized on the tonoplast, P2 was found both at the tonoplast and at other locations in the cell. As Brome mosaic virus replication complexes have been found to be associated with the endoplasmic reticulum (M. A. Restrepo-Hartwig and P. Ahlquist, J. Virol. 70:8908-8916, 1996), viruses in the family Bromoviridae apparently select different cellular membranes for the assembly of their replication complexes.

* Corresponding author. Mailing address: Institute of Molecular Plant Sciences, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Phone: (31)715274749. Fax: (31)715274469. E-mail: j.bol{at}chem.leidenuniv.nl.

Journal of Virology, February 2001, p. 1879-1887, Vol. 75, No. 4
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.4.1879-1887.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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