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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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