Alanine Scanning of Poliovirus 2C ^ATPase Reveals New Genetic Evidence that Capsid Protein/2C ^ATPase Interactions Are Essential for Morphogenesis

Abstract

<jats:p>Polypeptide 2C<jats:sup>ATPase</jats:sup>is one of the most thoroughly studied but least understood proteins in the life cycle of poliovirus. Within the protein, multiple functional domains important for uncoating, host cell membrane alterations, and RNA replication and encapsidation have previously been identified. In this study, charged to alanine-scanning mutagenesis was used to generate conditional-lethal mutations in hitherto-uncharacterized domains of the 2C<jats:sup>ATPase</jats:sup>polypeptide, particularly those involved in morphogenesis. Adjacent or clustered charged amino acids (2 to 4), scattered along the 2C<jats:sup>ATPase</jats:sup>coding sequence, were replaced with alanines. RNA transcripts of mutant poliovirus cDNA clones were transfected into HeLa cells. Subsequently, 10 lethal, 1 severely temperature-sensitive, 2 quasi-infectious, and 3 wild type-like mutants were identified. Using a luciferase-containing reporter virus, we demonstrated RNA replication defects in all lethal and quasi-infectious mutants. Temperature-sensitive mutants were defective in RNA replication only at the restricted temperatures. Furthermore, we characterized a quasi-infectious mutant (K<jats:sub>6</jats:sub>A/K<jats:sub>7</jats:sub>A) that produced a suppressor mutation (G<jats:sub>1</jats:sub>R) and a novel 2B∧2C<jats:sup>ATPase</jats:sup>cleavage site (Q∧R). Surprisingly, this cleavage site mutation did not interfere with normal processing of the polyprotein. These mutants have led to the identification of several new sites within the 2C<jats:sup>ATPase</jats:sup>polypeptide that are required for RNA replication. In addition, analysis of the suppressor mutants has revealed a new domain near the C terminus of 2C<jats:sup>ATPase</jats:sup>that is involved in encapsidation, possibly achieved through interaction with an amino acid sequence between NTP binding motifs A and B of 2C<jats:sup>ATPase</jats:sup>. Most importantly, the identification of suppressor mutations in both 2C<jats:sup>ATPase</jats:sup>and the capsid domains (VP1 and VP3) of poliovirus has confirmed that an interaction between 2C<jats:sup>ATPase</jats:sup>and capsid proteins is involved in viral morphogenesis.</jats:p>

Exchanges of genomic domains between poliovirus and other cocirculating species C enteroviruses reveal a high degree of plasticity

“…This protein is part of the replication complex, in which it acts as an NTPase, and it is also involved in viral morphogenesis24243. It contains RNA-binding domains but, to our knowledge, no direct interactions have been observed between this protein and the 5′UTR.…”

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

“…ATPase discriminates against even closely related CP precursors (45)(46)(47)(48). We suggest that C-cluster enteroviruses and possibly all viruses of the genus Enterovirus of Picornaviridae use protein/protein recognition for achieving specificity of assembly (Fig.…”

Emergency Services of Viral RNAs: Repair and Remodeling

“…Mutational alterations of amino acids (i.e., nonsynonymous nucleotide substitutions) do not necessarily result in changed fitness. Even substantial alterations of the chemical nature of mutated amino acids do not obligatorily cause appreciable phenotypic changes, as exemplified by the outcome for replacements of certain charged amino acids by alanine in the 2C protein of poliovirus (237) or for a Val-to-Arg replacement in the RdRP of mengovirus (238). Similarly, some mutations in the TGK peptide of the oriL-interacting motif of 3C pro do not exhibit any appreciable phenotypic alterations, at…”