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Mutation of Host dnaJ Homolog Inhibits Brome Mosaic Virus Negative-Strand RNA Synthesis

Yuriko Tomita,^1, Tomomitsu Mizuno,¹ Juana Díez,^2, Satoshi Naito,¹ Paul Ahlquist,^2,3 and Masayuki Ishikawa^1,2,4*

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589,¹ CREST, Japan Science and Technology Corporation, Kawaguchi City, Saitama 332-0012, Japan,⁴ Institute for Molecular Virology,² Howard Hughes Medical Institute, University of Wisconsin—Madison, Madison, Wisconsin 53706³

Received 30 August 2002/ Accepted 27 November 2002

The replication of positive-strand RNA viruses involves not only viral proteins but also multiple cellular proteins and intracellular membranes. In both plant cells and the yeast Saccharomyces cerevisiae, brome mosaic virus (BMV), a member of the alphavirus-like superfamily, replicates its RNA in endoplasmic reticulum (ER)-associated complexes containing viral 1a and 2a proteins. Prior to negative-strand RNA synthesis, 1a localizes to ER membranes and recruits both positive-strand BMV RNA templates and the polymerase-like 2a protein to ER membranes. Here, we show that BMV RNA replication in S. cerevisiae is markedly inhibited by a mutation in the host YDJ1 gene, which encodes a chaperone Ydj1p related to Escherichia coli DnaJ. In the ydj1 mutant, negative-strand RNA accumulation was inhibited even though 1a protein associated with membranes and the positive-strand RNA3 replication template and 2a protein were recruited to membranes as in wild-type cells. In addition, we found that in ydj1 mutant cells but not wild-type cells, a fraction of 2a protein accumulated in a membrane-free but insoluble, rapidly sedimenting form. These and other results show that Ydj1p is involved in forming BMV replication complexes active in negative-strand RNA synthesis and suggest that a chaperone system involving Ydj1p participates in 2a protein folding or assembly into the active replication complex.

Present address: Innovation Plaza Hokkaido, Japan Science and Technology Corporation, Sapporo 060-0819, Japan.

Present address: Universidad Pompeu Fabra, Área del Mar, Microbiology Unit, 08003 Barcelona, Spain.

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