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Journal of Virology, November 2002, p. 11321-11328, Vol. 76, No. 22
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.22.11321-11328.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Role of the Alfalfa Mosaic Virus Methyltransferase-Like Domain in Negative-Strand RNA Synthesis

A. Corina Vlot, Aymeric Menard,{dagger} and John F. Bol*

Gorlaeus Laboratories, Institute of Molecular Plant Sciences, Leiden University, Leiden, The Netherlands

Received 10 May 2002/ Accepted 15 August 2002

RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (AMV) encode the replicase proteins P1 and P2, respectively. P1 contains a methyltransferase-like domain in its N-terminal half, which has a putative role in capping the viral RNAs. Six residues in this domain that are highly conserved in the methyltransferase domains of alphavirus-like viruses were mutated individually in AMV P1. None of the mutants was infectious to plants. Mutant RNA 1 was coexpressed with wild-type (wt) RNAs 2 and 3 from transferred DNA vectors in Nicotiana benthamiana by agroinfiltration. Mutation of His-100 or Cys-189 in P1 reduced accumulation of negative- and positive-strand RNA in the infiltrated leaves to virtually undetectable levels. Mutation of Asp-154, Arg-157, Cys-182, or Tyr-266 in P1 reduced negative-strand RNA accumulation to levels ranging from 2 to 38% of those for the wt control, whereas positive-strand RNA accumulation by these mutants was 2% or less. The (transiently) expressed replicases of the six mutants were purified from the agroinfiltrated leaves. Polymerase activities of these preparations in vitro ranged from undetectable to wt levels. The data indicate that, in addition to its putative role in RNA capping, the methyltransferase-like domain of P1 has distinct roles in replication-associated functions required for negative-strand RNA synthesis. The defect in negative-strand RNA synthesis of the His-100 and Cys-189 mutants could be complemented in trans by coexpression of wt P1.

* Corresponding author. Mailing address: Institute of Molecular Plant Sciences, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands. Phone (31) 71-5274749. Fax: (31) 71-5274469. E-mail: j.bol{at}chem.leidenuniv.nl.

{dagger} Present address: Ecologie Microbienne, UMR 5557, Batiment Gregor Mendel, Université Claude-Bernard Lyon 1, 69622 Villeurbanne, France.

Journal of Virology, November 2002, p. 11321-11328, Vol. 76, No. 22
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.22.11321-11328.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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