NMR solution structure of poliovirus uridylyated peptide linked to the genome (VPgpU).
Picornaviruses have a 22-24 amino acid peptide, VPg, bound covalently at the 5' end of their RNA, that is essential for replication. VPgs are uridylylated at a conserved tyrosine to form VPgpU, the primer of RNA synthesis by the viral polymerase. This first complete structure for any uridylylated VPg, of poliovirus type 1 (PV1)-VPgpU, shows that conserved amino acids in VPg stabilize the bound UMP, with the uridine atoms involved in base pairing and chain elongation projected outward. Comparing this structure to PV1-VPg and partial structures of VPg/VPgpU from other picornaviruses suggests that enteroviral polymerases require a more stable VPg structure than does the distantly related aphthovirus, foot and mouth disease virus (FMDV). The glutamine residue at the C-terminus of PV1-VPgpU lies in back of the uridine base and may stabilize its position during chain elongation and/or contribute to base specificity. Under in vivo-like conditions with the authentic cre(2C) hairpin RNA and Mg(2+), 5-methylUTP cannot compete with UTP for VPg uridylyation in an in vitro uridylyation assay, but both nucleotides are equally incorporated by PV1-polymerase with Mn(2+) and a poly-A RNA template. This indicates the 5 position is recognized under in vivo conditions. The compact VPgpU structure docks within the active site cavity of the PV-polymerase, close to the position seen for the fragment of FMDV-VPgpU with its polymerase. This structure could aid in design of novel enterovirus inhibitors, and stabilization upon uridylylation may also be pertinent for post-translational uridylylation reactions that underlie other biological processes.
Schein
Catherine H
CH
Computational Biology, Sealy Center for Structural Biology and Molecular Biophysics, Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555-0857, USA. chschein@utmb.edu
Oezguen
Numan
N
van der Heden van Noort
Gerbrand J
GJ
Filippov
Dmitri V
DV
Paul
Aniko
A
Kumar
Eric
E
Braun
Werner
W
D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000737:chemistry; D001667:Binding, Competitive; D015195:Drug Design; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D017955:Poliovirus / Q000254:growth & development / Q000502:physiology*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D014500:Uracil Nucleotides / Q000737:chemistry* / Q000378:metabolism; D014542:Uridine Monophosphate / Q000737:chemistry / Q000378:metabolism; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D010455:Peptides; D014500:Uracil Nucleotides; C520979:VPg protein, poliovirus; D014764:Viral Proteins; D014542:Uridine Monophosphate; C090443:polymerase 3Dpol, poliovirus; D012324:RNA-Dependent RNA Polymerase; D014544:Uridine Triphosphate