Picornavirus genome replication. Identification of the surface of the poliovirus (PV) 3C dimer that interacts with PV 3Dpol during VPg uridylylation and construction of a structural model for the PV 3C2-3Dpol complex.
Picornaviruses have a peptide termed VPg covalently linked to the 5'-end of the genome. Attachment of VPg to the genome occurs in at least two steps. First, Tyr-3 of VPg, or some precursor thereof, is used as a primer by the viral RNA-dependent RNA polymerase, 3Dpol, to produce VPg-pUpU. Second, VPg-pUpU is used as a primer to produce full-length genomic RNA. Production of VPg-pUpU is templated by a single adenylate residue located in the loop of an RNA stem-loop structure termed oriI by using a slide-back mechanism. Recruitment of 3Dpol to and its stability on oriI have been suggested to require an interaction between the back of the thumb subdomain of 3Dpol and an undefined region of the 3C domain of viral protein 3CD. We have performed surface acidic-to-alanine-scanning mutagenesis of 3C to identify the surface of 3C with which 3Dpol interacts. This analysis identified numerous viable poliovirus mutants with reduced growth kinetics that correlated to reduced kinetics of RNA synthesis that was attributable to a change in VPg-pUpU production. Importantly, these 3C derivatives were all capable of binding to oriI as well as wild-type 3C. Synthetic lethality was observed for these mutants when placed in the context of a poliovirus mutant containing 3Dpol-R455A, a residue on the back of the thumb required for VPg uridylylation. These data were used to guide molecular docking of the structures for a poliovirus 3C dimer and 3Dpol, leading to a structural model for the 3C(2)-3Dpol complex that extrapolates well to all picornaviruses.
Shen
Miaoqing
M
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Reitman
Zachary J
ZJ
Zhao
Yan
Y
Moustafa
Ibrahim
I
Wang
Qixin
Q
Arnold
Jamie J
JJ
Pathak
Harsh B
HB
Cameron
Craig E
CE
D001483:Base Sequence; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D019281:Dimerization; D016679:Genome, Viral; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010849:Picornaviridae / Q000235:genetics* / Q000502:physiology; D017955:Poliovirus / Q000235:genetics* / Q000502:physiology; D011487:Protein Conformation; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics; D012997:Solvents; D014764:Viral Proteins / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics*
D017931:DNA Primers; D012261:Ribonucleoproteins; D012997:Solvents; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase