The GEF1 Proton-Chloride Exchanger Affects Tombusvirus Replication via Regulation of Copper Metabolism in Yeast

Replication 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 vitro activity of the purified tombusvirus replicase fromgef1 Δ yeast was low and that thein vitro assembly of the viral replicase in a cell extract was inhibited by the cytosolic fraction obtained fromgef1 Δ 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 vitro assembly of the viral replicase, on the activity of the viral RNA-dependent RNA polymerase, and an inhibitory effect of deletion ofCCC2 copper 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 benthamiana protoplasts 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.