title: Human rhinovirus type 14 gain-of-function mutants for oriI utilization define residues of 3C(D) and 3Dpol that contribute to assembly and stability of the picornavirus VPg uridylylation complex.
abstract: VPg linkage to the 5' ends of picornavirus RNAs requires production of VPg-pUpU. VPg-pUpU is templated by an RNA stem-loop (the cre or oriI) found at different locations in picornavirus genomes. At least one adaptive mutation is required for human rhinovirus type 14 (HRV-14) to use poliovirus type 3 (PV-3) or PV-1 oriI efficiently. One mutation changes Leu-94 of 3C to Pro; the other changes Asp-406 of 3Dpol to Asn. By using an in vitro VPg uridylylation system for HRV-14 that recapitulates biological phenotypes, we show that the 3C adaptive mutation functions at the level of 3C(D) and the 3D adaptive mutation functions at the level of 3Dpol. Pro-94 3C(D) has an expanded specificity and enhanced stability relative to wild-type 3C(D) that leads to production of more processive uridylylation complexes. PV-1/HRV-14 oriI chimeras reveal sequence specificity in 3C(D) recognition of oriI that resides in the upper stem. Asn-406 3Dpol is as active as wild-type 3Dpol in RNA-primed reactions but exhibits greater VPg uridylylation activity due to more efficient recruitment to and retention in the VPg uridylylation complex. Asn-406 3Dpol from PV-1 exhibits identical behavior. These studies suggest a two-step binding mechanism in the assembly of the 3C(D)-oriI complex that leads to unwinding of at least the upper stem of oriI and provide additional support for a direct interaction between the back of the thumb of 3Dpol and 3C that is required for 3Dpol recruitment to and retention in the uridylylation complex.
authors.affiliation: Pennsylvania State University, Department of Biochemistry and Molecular Biology, 201 Althouse Laboratory, University Park, PA 16802, USA.
authors.forename: Miaoqing
authors.initials: M
authors.lastname: Shen
authors.forename: Qixin
authors.initials: Q
authors.lastname: Wang
authors.forename: Yan
authors.initials: Y
authors.lastname: Yang
authors.forename: Harsh B
authors.initials: HB
authors.lastname: Pathak
authors.forename: Jamie J
authors.initials: JJ
authors.lastname: Arnold
authors.forename: Christian
authors.initials: C
authors.lastname: Castro
authors.forename: Stanley M
authors.initials: SM
authors.lastname: Lemon
authors.forename: Craig E
authors.initials: CE
authors.lastname: Cameron
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D016679:Genome, Viral; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010849:Picornaviridae / Q000235:genetics / Q000502:physiology; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D018741:Replication Origin; D012229:Rhinovirus / Q000235:genetics / Q000502:physiology*; D014542:Uridine Monophosphate / Q000378:metabolism; D014758:Viral Core Proteins / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics*
chemical_list: D012367:RNA, Viral; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D014542:Uridine Monophosphate; D012321:DNA-Directed RNA Polymerases