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

Abstract

HBB [2-(α-hydroxybenzyl)-benzimidazole] and guanidine are potent inhibitors of picornavirus replication. Among other evidence, limited cross-resistance and a synergistic effect of both inhibitors suggest similar but not identical mechanisms of antiviral action. Echovirus-9 variants resistant to each of these drugs were characterized and sequenced. Complete resistance to HBB or guanidine was shown to be due to single but different point mutations in the non-structural protein 2C. Protein 2C was expressed as GST fusion and His-tagged proteins for the wild-type and various mutants. Although three mutations were located in or near conserved NTP binding motifs, NTPase activity was not altered in the presence of HBB or guanidine.

The Thiazolobenzimidazole TBZE-029 Inhibits Enterovirus Replication by Targeting a Short Region Immediately Downstream from Motif C in the Nonstructural Protein 2C

“…Besides these "main" mutations, guanidine-resistant poliovirus was shown to carry some additional mutations that were located at residue 143, 227, or 233. It should be emphasized that residue 227 is also involved in the resistance of echovirus to HBB (39) and, as reported here, of coxsackievirus to TBZE-029. Shimizu et al (61) demonstrated that poliovirus carrying either of the guanidine "main" mutations was not only resistant to guanidine but also cross-resistant to MRL-1237.…”

“…Reintroduction of these mutations, either alone or combined, into a full-length clone of CVB3 generated seven recombinant clones, which were further characterized. Single amino acid mutations at positions Interestingly, previous studies with the antiviral compound HBB in echovirus 9 reported that altered sensitivity to this drug mapped, similarly to our findings, to the Ile and Ala residues at positions 227 and 229, respectively (32,39). In particular, the I227L mutation, either alone or combined with the A229V mutation, conferred resistance to HBB.…”

“…In particular, the I227L mutation, either alone or combined with the A229V mutation, conferred resistance to HBB. Akin to the observations in the current study, a single mutation of Ala at position 229, however, led to a noninfectious RNA transcript, which could be rescued by the addition of HBB or GuaHCl (32,39). Studies of cross-resistance to HBB, using the recombinant TBZE-029-resistant coxsackievirus clones, showed an identical pattern of resistance (Table 2).…”

“…Bienz and coworkers showed that guanidine prevents the association of 2BC with host membrane structures during viral replication (13). Moreover, it was shown that guanidine at millimolar concentrations inhibits ATP hydrolysis (51), although other studies were not able to confirm this observation (39,57). Another compound that has been known for decades and that selectively blocks enterovirus replication is 2-(alpha-hydroxybenzyl)-benzimidazole (HBB) (24)(25)(26)(27) (Fig.…”

“…1). Resistance to (or dependence on) HBB of echovirus is caused by single amino acid mutations in protein 2C (32,39). Also, compound 1-(4-fluorophenyl)-2-[(4-imino-1,4-dihydropyridin-1-yl)methyl]benzimidazole hydrochloride (MRL-1237) (Fig.…”