Positive-strand RNA viruses build viral replication organelles (VROs) with the help of co-opted host factors. The biogenesis of the membranous VROs requires major metabolic changes in infected cells. Previous studies showed that tomato bushy stunt virus (TBSV) hijacks several glycolytic enzymes to produce ATP locally within VROs. In this work, we demonstrate that the yeast Pfk2p phosphofructokinase, which performs a rate-limiting and highly regulated step in glycolysis, interacts with the TBSV p33 replication protein. Deletion of PFK2 reduced TBSV replication in yeast, suggesting proviral role for Pfk2p. TBSV also co-opted two plant phosphofructokinases, which supported viral replication and ATP production within VROs, thus acting as proviral factors. Three other phosphofructokinases inhibited TBSV replication and they reduced ATP production within VROs, thus functioning as antiviral factors. Altogether, different phosphofructokinases have proviral or antiviral roles. This suggests on-going arms race between tombusviruses and their hosts to control glycolysis pathway in infected cells.