Attenuating Mutations in Coxsackievirus B3 Map to a Conformational Epitope That Comprises the Puff Region of VP2 and the Knob of VP3

Abstract

Ten antibody escape mutants of coxsackievirus B3 (CVB3) were used to identify nucleotide substitutions that determine viral virulence for the heart and pancreas. The P1 region, encoding the structural genes of each mutant, was sequenced to identify mutations associated with the lack of neutralization. Eight mutants were found to have a lysine-to arginine mutation in the puff region of VP2, while two had a glutamate-to-glycine substitution in the knob of VP3. Two mutants, EM1 and EM10, representing each of these mutations, were further analyzed, initially by determining their entire sequence. In addition to the mutations in P1, EM1 was found to have two mutations in the 3D polymerase, while EM10 had a mutation in stem-loop II of the 5 nontranslated region (5NTR). The pathogenesis of the mutants relative to that of CVB3 strain RK [CVB3(RK)] then was examined in A/J mice. Both mutants were found to be less cardiotropic than the parental strain, with a 40-fold (EM1) or a 100-to 1,000-fold (EM10) reduction in viral titers in the heart relative to the titers of CVB3(RK). The mutations in VP2, VP3, and the 5NTR were introduced independently into the RK infectious clone, and the phenotypes of the progeny viruses were determined. The results substantiated that the VP2 and VP3 mutations reduced cardiovirulence, while the 5NTR mutation in EM10 was associated with a more virulent phenotype when expressed on its own. Stereographic imaging of the two mutations in the capsomer showed that they lie in close proximity on either side of a narrow cleft between the puff and the knob, forming a conformational epitope that is part of the putative binding site for coreceptor DAF.

Complex genetic control of host susceptibility to coxsackievirus B3-induced myocarditis

“…13 Remarkably, many aspects of viral myocarditis may be recapitulated in experimental autoimmune models as induced by cardiac myosin and an adjuvant. 14 Pathological outcome of CVB3-induced myocarditis is determined by a complex interplay between viral [15][16][17][18] and host factors. At least two host proteins, decay accelerating factor (DAF) 19 and coxsackievirus-adenovirus receptor (CAR), 20,21 function as receptors for virus attachment and entry through binding with virus capsid proteins.…”

Molecular characterization of three enteroviral strains isolated in Kuwait from young children with serious conditions

“…Like the Puff region of VP2, the Knob region of VP3 is known to contain a major neutralization site for both poliovirus and rhinovirus [30,32]. Furthermore, mutations in the VP3 Knob region have been suggested to have a role in the CVB3-induced cardiovirulence [31]. Whether or not the nonsynonymous mutations detected in the capsid region of the three clinical isolates play a role in virulence and pathogenesis of enterovirus infection, needs further investigation.…”

“…The three clinical isolates in this study had amino acid substitutions in the Puff region of the VP2 protein. Amino acid substitutions in the Puff region of CVB3 VP2 protein were previously linked to cardiovirulence [31]. Like the Puff region of VP2, the Knob region of VP3 is known to contain a major neutralization site for both poliovirus and rhinovirus [30,32].…”

A Genetically Engineered Attenuated Coxsackievirus B3 Strain Protects Mice against Lethal Infection

“…The construction of the infectious clones pCVB3(KR) and pCVB3(EG) was previously described (26). The plasmid pCVB3(KR) has a single mutation of A to G at nucleotide 1421, resulting in a lysine (K)-to-arginine (R) substitution at amino acid 158 of the capsid protein VP2 (K2158R), while pCVB3(EG) contains a single mutation of A to G at nucleotide 1916, resulting in a glutamic acid (E)-to-glycine (G) substitution at amino acid 60 of VP3 (E3060G).…”

“…In 1996, a single amino acid substitution in the puff region of VP2 (asparagine to aspartate at amino acid 165) was found to reduce the myocardicity of a CVB3 mutant, H310A1 (14). Recently, we identified two mutations in the capsid proteins that affected cardiovirulence in studies of a panel of "escape" mutants (EMs 1 to 10) derived by treating a parental myocarditic strain, CVB3(RK), with a highly neutralizing monoclonal antibody (26). Two mutations were identified that were associated with both a lack of neutralization by the monoclonal antibody and reduced cardiovirulence.…”