The GEF1 Proton-Chloride Exchanger Affects Tombusvirus Replication via Regulation of Copper Metabolism in Yeast
ABSTRACTReplication of plus-strand RNA viruses [(+)RNA viruses] is performed by viral replicases, whose function is affected by many cellular factors in infected cells. In this paper, we demonstrate a surprising role for Gef1p proton-chloride exchanger in replication ofTomato bushy stunt virus(TBSV) model (+)RNA virus. A genetic approach revealed that Gef1p, which is the only proton-chloride exchanger inSaccharomyces cerevisiae, is required for TBSV replication in the yeast model host. We also show that thein vitroactivity of the purified tombusvirus replicase fromgef1Δ yeast was low and that thein vitroassembly of the viral replicase in a cell extract was inhibited by the cytosolic fraction obtained fromgef1Δ yeast. Altogether, our data reveal that Gef1p modulates TBSV replication via regulating Cu2+metabolism in the cell. This conclusion is supported by several lines of evidence, including the direct inhibitory effect of Cu2+ions on thein vitroassembly of the viral replicase, on the activity of the viral RNA-dependent RNA polymerase, and an inhibitory effect of deletion ofCCC2copper pump on TBSV replication in yeast, while altered iron metabolism did not reduce TBSV replication. In addition, applying a chloride channel blocker impeded TBSV replication inNicotiana benthamianaprotoplasts or in whole plants. Overall, blocking Gef1p function seems to inhibit TBSV replication through altering Cu2+ion metabolism in the cytosol, which then inhibits the normal functions of the viral replicase.