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

Abstract

Protein 2C(ATPase) of picornaviruses is involved in the rearrangement of host cell organelles, viral RNA replication, and encapsidation. However, the biochemical and molecular mechanisms by which 2C(ATPase) engages in these processes are not known. To characterize functional domains of 2C(ATPase), we have focused on a cysteine-rich motif near the carboxy terminus of poliovirus 2C(ATPase). 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. 2C(ATPase) as well as its precursors 2BC and P2 were found to exist in a reduced form in poliovirus-infected cells.

FIG. 1

General features of poliovirus protein 2C^ATPase. Regions of protein 2C^ATPase with suggested functions are indicated by brackets and amino acid positions. A, B, and C denote motifs conserved among superfamily 3 helicases and are required for the ATPase activity (44). Black diamonds indicate locations of previously published mutations that affect RNA replication; white diamonds indicate locations of hallmark mutations found in most guanidine-resistant and -dependent virus mutants (58). Arrows delineate a predicted domain organization (57).

FIG. 2

Protein sequence alignment of the cysteine-rich motif in protein 2C^ATPase of enteroviruses and rhinoviruses. Cysteine residues are printed in bold. PCSs are shaded and numbered from left to right. Abbreviations: PV, poliovirus; EV, bovine enterovirus; CAV and CBV, coxsackie A virus and coxsackie B virus, respectively; SVDV, swine vesicular disease virus; HRV, human rhinovirus. a, numbering of amino acid residues according to the PVM sequence.

FIG. 3

In vitro translation reactions in the absence of RNA (lane 1) or primed with transcript RNA of pT7PVM (lane 2), pT7PVMΔPCS1 (lane 3), or pT7PVMΔPCS2 (lane 4). An extract of poliovirus-infected HeLa cells was used as marker (lane M). Viral proteins were labeled with [³⁵S]methionine and visualized by autoradiography. Positions of nonstructural proteins are indicated to the left.

FIG. 4

Single-cycle virus growth curves. HeLa cell monolayers were infected at an MOI of 10 PFU/cell. At different time points p.i., supernatants were harvested and their virus titers were determined.

FIG. 5

In vivo labeling of viral proteins with [³⁵S]methionine. (A) Viral proteins were labeled in the presence of guanidine-HCl at the time period and temperature indicated. TCA-precipitable radioactivity was measured by liquid scintillation counting. (B) Viral proteins were labeled in the absence of guanidine-HCl from 4 to 5.5 h p.i. Proteins were separated by SDS-PAGE and visualized by autoradiography. Positions of nonstructural proteins are indicated.

FIG. 6

In vivo labeling of viral RNA with [³H]uridine starting at 1.75 h p.i. Cells were lysed at different time points, and TCA-precipitable radioactivity was measured by liquid scintillation counting.

FIG. 7

Proteins 2C^ATPase, 2BC, and P2 are in a reduced form in infected cells. Poliovirus-infected cells were lysed in the absence (−) or presence (+) of 10 mM IAM. Proteins were separated by SDS-PAGE in the absence (−) or presence (+) of 10 mM DTT. 2C^ATPase, 2BC, and P2 were visualized by Western blotting using an anti-2C monoclonal antibody.

FIG. 8

The cysteine-rich motif binds zinc in vitro. (A) Amino acid sequences of peptides expressed as GST fusion proteins in E. coli. GST-CR^wt contained residues 255 to 297 of poliovirus 2C^ATPase encompassing the cysteine-rich motif, residues 269 to 286. In GST-CR^mut, the cysteine and histidine residues were replaced by serine and glutamine, respectively. GST was the product of the expression plasmid pGEX-KG. (B) Flowchart of GST-CR^wt purification and treatment with zinc acetate and/or EDTA. The block diagram (bottom) shows the metal content of four protein preparations, determined by measuring the absorption at 490 nm in the presence of 0.1 mM PAR. (C) Typical standard curve showing the relationship between zinc concentration and absorption in the presence of PAR. The best-fit curve and its correlation were calculated with the program Cricket Graph (Cricket Software, Malvern, Pa.). (D) Metal content of GST-CR^wt, GST-CR^mut, and GST after zinc treatment and purification. The average and standard deviation of quadruplicate measurements are shown.

FIG. 8

The cysteine-rich motif binds zinc in vitro. (A) Amino acid sequences of peptides expressed as GST fusion proteins in E. coli. GST-CR^wt contained residues 255 to 297 of poliovirus 2C^ATPase encompassing the cysteine-rich motif, residues 269 to 286. In GST-CR^mut, the cysteine and histidine residues were replaced by serine and glutamine, respectively. GST was the product of the expression plasmid pGEX-KG. (B) Flowchart of GST-CR^wt purification and treatment with zinc acetate and/or EDTA. The block diagram (bottom) shows the metal content of four protein preparations, determined by measuring the absorption at 490 nm in the presence of 0.1 mM PAR. (C) Typical standard curve showing the relationship between zinc concentration and absorption in the presence of PAR. The best-fit curve and its correlation were calculated with the program Cricket Graph (Cricket Software, Malvern, Pa.). (D) Metal content of GST-CR^wt, GST-CR^mut, and GST after zinc treatment and purification. The average and standard deviation of quadruplicate measurements are shown.

FIG. 8

The cysteine-rich motif binds zinc in vitro. (A) Amino acid sequences of peptides expressed as GST fusion proteins in E. coli. GST-CR^wt contained residues 255 to 297 of poliovirus 2C^ATPase encompassing the cysteine-rich motif, residues 269 to 286. In GST-CR^mut, the cysteine and histidine residues were replaced by serine and glutamine, respectively. GST was the product of the expression plasmid pGEX-KG. (B) Flowchart of GST-CR^wt purification and treatment with zinc acetate and/or EDTA. The block diagram (bottom) shows the metal content of four protein preparations, determined by measuring the absorption at 490 nm in the presence of 0.1 mM PAR. (C) Typical standard curve showing the relationship between zinc concentration and absorption in the presence of PAR. The best-fit curve and its correlation were calculated with the program Cricket Graph (Cricket Software, Malvern, Pa.). (D) Metal content of GST-CR^wt, GST-CR^mut, and GST after zinc treatment and purification. The average and standard deviation of quadruplicate measurements are shown.

FIG. 9

Metal content and enzymatic activity of EDTA- and zinc-treated GST-2C. Bacterially expressed GST-2C was bound to glutathione-Sepharose beads in the presence of 1 mM EDTA or 0.1 mM zinc acetate. The beads were washed four times with 100 bead volumes each and eluted. (A) Metal content of the differently treated GST-2C preparations. (B) Specific ATPase activity of the two preparations. Inorganic phosphate released from ATP was determined by a colorimetric assay (44). The average and standard deviation of triplicate measurements are shown.