Mapping genetic determinants of the cell-culture growth phenotype of enterovirus 71

Enterovirus 71 (EV71) is a member of the species Human enterovirus A within the family Picornaviridae and is a major causative agent of epidemics of hand, foot and mouth disease associated with severe neurological disease. Three EV71 genogroups, designated A, B and C, have been identified, with 75–84 % nucleotide sequence similarity between them. Two strains, EV71-26M (genogroup B) and EV71-6F (genogroup C), were found to have distinct cell-culture growth (26M, rapid; 6F, slow) and plaque-formation (26M, large; 6F, small) phenotypes. To identify the genome regions responsible for the growth phenotypes of the two strains, a series of chimeric viruses was constructed by exchanging the 5′ untranslated region (UTR), P1 structural protein or P2/P3 non-structural protein gene regions plus the 3′UTR using infectious cDNA clones of both virus strains. Analysis of reciprocal virus chimeras revealed that the 5′UTRs of both strains were compatible, but not responsible for the observed phenotypes. Introduction of the EV71-6F P1 region into the EV71-26M clone resulted in a small-plaque and slow-growth phenotype similar to that of EV71-6F, whereas the reciprocal chimera displayed intermediate-growth and intermediate-sized plaque phenotypes. Introduction of the EV71-26M P2–P3–3′UTR regions into the EV71-6F clone resulted in a large-plaque and rapid-growth phenotype identical to that of strain EV71-26M, whereas the reciprocal chimera retained the background strain large-plaque phenotype. These results indicate that, although both the P1 and P2–P3–3′UTR genome regions influence the EV71 growth phenotype in cell culture, phenotype expression is dependent on specific genome-segment combinations and is not reciprocal

Direct Interaction between Two Viral Proteins, the Nonstructural Protein 2CATPase and the Capsid Protein VP3, Is Required for Enterovirus Morphogenesis

In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV), is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2CATPase in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel “reporter virus”, we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20) and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2CATPase of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2CATPase and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20) were blocked in encapsidation (no virus after blind passages) but could be rescued if the capsid and 2CATPase coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i) genome replication is known to be stringently linked to translation, (ii) morphogenesis is known to be stringently linked to genome replication, (iii) newly synthesized 2CATPase is an essential component of the replication complex, and (iv) 2CATPase has specific affinity to capsid protein(s). These conditions lead to morphogenesis at the site where newly synthesized genomes emerge from the replication complex

Similar interactions of the poliovirus and rhinovirus 3D polymerases with the 3 ' untranslated region of rhinovirus 14

We showed previously that a human rhinovirus 14 (HRV14) 3' untranslated region (3' UTR) on a poliovirus genome was able to replicate with nearly wild-type kinetics (J. B. Rohll, D. H. Moon, D. J. Evans, and J. W. Almond, J, Virol 69:7835-7844, 1995). This enabled the HRV14 single 3' UTR stem-loop structure to be studied in combination with a sensitive reporter system, poliovirus FLC/REP, in which the capsid coding region is replaced by an in-frame chloramphemicol acetyltransferase (CAT) gene. Using such a construct, we identified a mutant (designated mut4), in which the structure and stability of the stem were predicted to be maintained, that replicated very poorly as determined by its level of CAT activity. The effect of this mutant 3' UTR on replication has been further investigated by transferring it onto the full-length cDNAs of both poliovirus type 3 (PV3) and HRV14. Virus was recovered with a parental plaque phenotype at a low frequency, indicating the acquisition of compensating changes, which sequence analysis revealed were, in both poliovirus- and rhinovirus-derived viruses, located in the active-site cleft of 3D polymerase and involved the substitution of Asn18 for Tyr. These results provide further evidence of a specific interaction between the 3' UTR of picornaviruses and the viral polymerase and also indicate similar interactions of the 3' UTR of rhinovirus with both poliovirus and rhinovirus polymerases.</p

Similar interactions of the poliovirus and rhinovirus 3D polymerases with the 3 ' untranslated region of rhinovirus 14

We showed previously that a human rhinovirus 14 (HRV14) 3' untranslated region (3' UTR) on a poliovirus genome was able to replicate with nearly wild-type kinetics (J. B. Rohll, D. H. Moon, D. J. Evans, and J. W. Almond, J, Virol 69:7835-7844, 1995). This enabled the HRV14 single 3' UTR stem-loop structure to be studied in combination with a sensitive reporter system, poliovirus FLC/REP, in which the capsid coding region is replaced by an in-frame chloramphemicol acetyltransferase (CAT) gene. Using such a construct, we identified a mutant (designated mut4), in which the structure and stability of the stem were predicted to be maintained, that replicated very poorly as determined by its level of CAT activity. The effect of this mutant 3' UTR on replication has been further investigated by transferring it onto the full-length cDNAs of both poliovirus type 3 (PV3) and HRV14. Virus was recovered with a parental plaque phenotype at a low frequency, indicating the acquisition of compensating changes, which sequence analysis revealed were, in both poliovirus- and rhinovirus-derived viruses, located in the active-site cleft of 3D polymerase and involved the substitution of Asn18 for Tyr. These results provide further evidence of a specific interaction between the 3' UTR of picornaviruses and the viral polymerase and also indicate similar interactions of the 3' UTR of rhinovirus with both poliovirus and rhinovirus polymerases.</p