NMR solution structure of poliovirus uridylyated peptide linked to the genome (VPgpU)

Abstract

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 ++ , 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 ++ 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.

Initiation of protein-primed picornavirus RNA synthesis

“…The structure consists of a long loop (residues 1-14) and a short C-terminal helix. An NMR structure of chemically synthesized VPgpU was more stable than that of free VPg and exhibited a defined globular structure (135). FMDV is unique in that it encodes 3 distinct VPg sequences all of which function as substrates in vitro for uridylylation and all of them can be observed attached to viral RNAs (71, 100).…”

Structures of the Compact Helical Core Domains of Feline Calicivirus and Murine Norovirus VPg Proteins

“…The solution structures of the 22-amino-acid VPg from poliovirus, a picornavirus, have been determined using nuclear magnetic resonance (NMR) spectroscopy for both the native peptide and VPg that has been nucleotidylated on its acceptor Tyr 3 residue (48,49). In its native form, poliovirus VPg appears to be highly flexible; a defined conformation was observed only in the presence of high concentrations (1 M) of the organic solvent trimethylamine N-oxide (TMAO).…”

“…In its native form, poliovirus VPg appears to be highly flexible; a defined conformation was observed only in the presence of high concentrations (1 M) of the organic solvent trimethylamine N-oxide (TMAO). Intriguingly, the nucleotide-linked poliovirus VPg-pU appeared more stable and exhibited a defined, globular conformation in an aqueous solution that lacked TMAO (48). However, the physiological relevance of this structure is uncertain; although the poliovirus VPg-pU structure may be computationally docked with the viral three-dimensional (3D) polymerase, the resulting model did not position the uridine in VPg close enough to the active-site residues of the polymerase to plausibly account for the nucleotidylation reaction.…”

Sequence specificity for uridylylation of the viral peptide linked to the genome (VPg) of enteroviruses

“…Synthetic VPgpU, used for calibrating the position of VPgpU on gels in early stages of this work, was generated as described previously (Schein et al, 2010; van der Heden van Noort et al, 2013). VPgs were dissolved in water and their concentration determined using the extinction coefficients for tyrosine (the only UV-absorbing amino acid in the peptide) in the range of 220-280 nm.…”

“…VPg, before uridylylation, in solution has a flexible, or even disordered structure (Schein et al, 2006b), which might also be stabilized by binding to cellular components or the polymerase. In contrast, chemically synthesized, uridylylated PV-VPgpU has a very stable structure in solution (Schein et al, 2010). The NMR structure indicated that the positively charged residues directly coordinate with the UMP moiety of the modified tyrosine.…”