Characterization of the Nucleoside Triphosphatase Activity of Poliovirus Protein 2C Reveals a Mechanism by Which Guanidine Inhibits Poliovirus Replication

Abstract

The highly conserved non-structural protein 2C of picornaviruses is involved in viral genome replication and encapsidation and in the rearrangement of intracellular structures. 2C binds RNA, has nucleoside triphosphatase activity, and shares three motifs with superfamily III helicases. Motifs "A" and "B" are involved in nucleotide triphosphate (NTP) binding and hydrolysis, whereas a function for motif "C" has not yet been demonstrated. Poliovirus RNA replication is inhibited by millimolar concentrations of guanidine hydrochloride (GdnHCl). Resistance and dependence to GdnHCl map to 2C. To characterize the nucleoside triphosphatase activity of 2C, we purified poliovirus recombinant 2C fused to glutathione S-transferase (GST-2C) from Escherichia coli. GST-2C hydrolyzed ATP with a K m of 0.7 mM. Other NTPs, including GTP, competed with ATP for binding to 2C but were poor substrates for hydrolysis. Mutation of conserved residues in motif A and B abolished ATPase activity, as did mutation of the conserved asparagine residue in motif C, an observation indicating the involvement of this motif in ATP hydrolysis. GdnHCl at millimolar concentrations inhibited ATP hydrolysis. Mutations in 2C that confer poliovirus resistant to or dependent on GdnHCl increased the tolerance to GdnHCl up to 100-fold.

Characterization of pharmacologically active compounds that inhibit poliovirus and enterovirus 71 infectivity

“…Identified mutations were located in the C terminus portion of protein 2C (amino acid positions 325 and 324 for metrifudil and N 6 -benzyladenosine resistance, respectively), which is responsible for the appearance of altered membranes in the PV-infected cells by unknown mechanism (Teterina et al, 1997). Mutations in this region were not observed in PV mutants resistant to guanidine hydrochloride, MRL-1237 and hydantoin, which suppress PV replication by inhibiting ATPase activity and/or encapsidation activity of protein 2C (Pfister & Wimmer, 1999;Shimizu et al, 2000;Vance et al, 1997). Therefore, the mechanisms of inhibition by metrifudil and N 6 -benzyladenosine via EV71 protein 2C remain to be further studied.…”

Dynamics of picornavirus RNA replication within infected cells

“…Using recombinant fusion proteins it has been shown that 2C has ATPase activity and that this activity of PV 2C is inhibited by low concentrations of guanidine (Pfister & Wimmer, 1999); however, this was not the case for the echovirus 9 2C protein (Klein et al, 2000). Many of the mutations in 2C that confer resistance to guanidine are in, or close to, the Walker motifs Tolskaya et al, 1994;Klein et al, 2000) but the single amino acid substitution reported in a single guanidine-resistant mutant of FMDV (strain O 6 ) was not near these motifs but close to the C terminus (Saunders et al, 1985).…”

The picornavirus replication inhibitors HBB and guanidine in the echovirus-9 system: the significance of viral protein 2C

“…It has been shown that this protein plays an important role in RNA replication, and the presence of 2C in enterovirus replication complexes comprised of membrane particles, viral RNA and other virus-encoded proteins has been demonstrated (Bienz et al, 1990). Functions like membrane binding, protein-protein interactions, NTPase activity and RNA binding have been revealed for poliovirus 2C by different in vitro techniques (Cuconati et al, 1998 ;Echeverri & Dasgupta, 1995 ;Mirzayan & Wimmer, 1994 ;Pfister & Wimmer, 1999 ;Rodriguez & Carrasco, 1993. NTPase activity was also observed in E9 (Klein et al, 1999).…”