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Journal of Virology, January 2000, p. 334-343, Vol. 74, No. 1
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Cysteine-Rich Motif in Poliovirus Protein 2CATPase Is Involved in RNA Replication and Binds Zinc In Vitro

Thomas Pfister,1 Keith W. Jones,2 and Eckard Wimmer1,*

Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794-5222,1 and Department of Applied Science, Brookhaven National Laboratory, Upton, New York 11973-50002

Received 22 April 1999/Accepted 30 September 1999

Protein 2CATPase of picornaviruses is involved in the rearrangement of host cell organelles, viral RNA replication, and encapsidation. However, the biochemical and molecular mechanisms by which 2CATPase engages in these processes are not known. To characterize functional domains of 2CATPase, we have focused on a cysteine-rich motif near the carboxy terminus of poliovirus 2CATPase. This region, which is well conserved among enteroviruses and rhinoviruses displaying an amino acid arrangement resembling zinc finger motifs, was studied by genetic and biochemical analyses. A mutation that replaced 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 replication was inhibited whereas translation and polyprotein processing, assayed in vitro and in vivo, appeared to be normal. An intragenomic second-site revertant which reinserted the missing coordination site for zinc and recovered RNA replication at the restrictive temperature was isolated. The cysteine-rich motif was sufficient to bind zinc in vitro, as assessed in the presence of 4-(2-pyridylazo)resorcinol by a colorimetric assay. Zinc binding, however, was not required for hydrolysis of ATP. 2CATPase as well as its precursors 2BC and P2 were found to exist in a reduced form in poliovirus-infected cells.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, NY 11794-5222. Phone: (516) 632-8787. Fax: (516) 632-8891. E-mail: wimmer{at}asterix.bio.sunysb.edu.

Journal of Virology, January 2000, p. 334-343, Vol. 74, No. 1
0022-538X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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