A cysteine-rich motif in poliovirus protein 2C(ATPase) is involved in RNA replication and binds zinc in vitro

Protein 2C(ATPase) of picornaviruses is involved in the rearrangement of ho st cell organelles, viral RNA replication, and encapsidation. However, the biochemical and molecular mechanisms by which 2C(ATPase) engages in these p rocesses are not known. To characterize functional domains of 2C(ATPase) we have focused on a cysteine-rich motif near the carboxy terminus of poliovi rus 2C(ATPase). This region, which is well conserved among enteroviruses an d rhinoviruses displaying an amino acid arrangement resembling zinc finger motifs, was studied by genetic and biochemical analyses. A mutation that re placed the first cysteine residue of the motif with a serine was lethal. A mutant virus which, lacked the second of four potential coordination sites for zinc was temperature sensitive, At the restrictive temperature, RNA rep lication was inhibited,whereas translation and polyprotein processing, assa yed in vitro and in vivo, appeared to be normal. An intragenomic second-sit e revertant which reinserted the missing coordination site for zinc and rec overed RNA replication at the restrictive temperature was isolated. The cys teine-rich motif was sufficient to bind zinc in vitro, as assessed in the p resence of 4-(2-pyridylazo)resorcinol by a colorimetric assay. Zinc binding , however, was not required for hydrolysis of ATP. 2C(ATPase) as web as its precursors 2BC and P2 were found to exist in a reduced form in poliovirus- infected cells.