Ribonucleotide reductase (RR) is essential to genome replication of mammals and the large DNA viruses that they host (pox, herpes, and adenoviruses). The mammalian and viral RR proteins are composed of an R1 subunit that contains the active site, and an R2 subunit that contains a diferric cluster and a spatially adjacent tyrosinate radical center. While not directly involved in the reduction of ribonucleotides to deoxyribonucleotides, this prosthetic group is essential to enzyme activity. Although the in vitro chemistry of the assembly of the diferric core has been studied in some detail, little is known about the cellular targeting of iron to the apoR2 protein. Furthermore, the dependence that RR has on cell iron, irrespective of the mechanism by which iron is trafficked to apoR2, suggests that the iron nutritional status of the cell will be a factor in the efficiency of this process. The likely source of iron for 112 is the cytosolic labile iron pool (LIP). This pool is sampled by membrane permeant chelators, the iron response element binding protein (IRP1), and apoferritin. We have shown that removing iron from this pool by either chelation or over-production of ferritin inhibits activation of viral RR, blocks the replication of viral DNA and suppresses the virulence of a DNA virus. This suggests that iron nutrition; iron trafficking and iron chelation will all impact on viral pathogenesis due to their effects on RR activation. This application describes experiments that evolve from test-tube to cultured cells to rodents. These experiments will evaluate the delivery of iron from the LIP to apoR2; to determine whether nutritional and genetic manipulation of this chelator-accessible intracellular pool suppresses a productive viral infection in cultured cells; and to determine whether complementary nutritional and genetic manipulation of mice and rats suppresses the virulence of a DNA virus in an in vivo model of viral pathogenesis. This work should provide new insight into the assembly of a critical iron prosthetic group in vivo and evidence that this process can be a target for intervention in a viral infection.
