title: Alfalfa mosaic virus replicase proteins P1 and P2 interact and colocalize at the vacuolar membrane.
abstract: 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.
authors.affiliation: Institute of Molecular Plant Sciences, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, The Netherlands.
authors.forename: M W
authors.initials: MW
authors.lastname: Van Der Heijden
authors.forename: J E
authors.initials: JE
authors.lastname: Carette
authors.forename: P J
authors.initials: PJ
authors.lastname: Reinhoud
authors.forename: A
authors.initials: A
authors.lastname: Haegi
authors.forename: J F
authors.initials: JF
authors.lastname: Bol
mesh_terms: D017794:Alfalfa mosaic virus / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D007425:Intracellular Membranes / Q000201:enzymology* / Q000821:virology; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D010944:Plants / Q000821:virology; D011233:Precipitin Tests; D011523:Protoplasts / Q000821:virology; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D014617:Vacuoles / Q000201:enzymology* / Q000821:virology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D014764:Viral Proteins; D008780:Methyltransferases; D012324:RNA-Dependent RNA Polymerase; D020365:RNA Helicases