Direct Inhibition of Tombusvirus Plus-Strand RNA Synthesis by a Dominant Negative Mutant of a Host Metabolic Enzyme, Glyceraldehyde-3-Phosphate Dehydrogenase, in Yeast and Plants

The replication of plus-strand RNA viruses depends on many cellular factors. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an abundant metabolic enzyme that is recruited to the replicase complex ofTomato bushy stunt virus (TBSV) and affects asymmetric viral RNA synthesis. To further our understanding on the role of GAPDH in TBSV replication, we used anin vitro TBSV replication assay based on recombinant p33 and p92pol viral replication proteins and cell-free yeast extract. We found that the addition of purified recombinant GAPDH to the cell extract prepared from GAPDH-depleted yeast results in increased plus-strand RNA synthesis and asymmetric production of viral RNAs. Our data also demonstrate that GAPDH interacts with p92pol viral replication protein, which may facilitate the recruitment of GAPDH into the viral replicase complex in the yeast model host. In addition, we have identified a dominant negative mutant of GAPDH, which inhibits RNA synthesis and RNA recruitmentin vitro . Moreover, this mutant also exhibits strong suppression of tombusvirus accumulation in yeast and in virus-infectedNicotiana benthamiana . Overall, the obtained data support the model that the co-opted GAPDH plays a direct role in TBSV replication by stimulating plus-strand synthesis by the viral replicase.