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(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.

Figure 1. Alignment of the sequence of PV-1 VPg (used in this study) with VPgs from other enteroviruses

Coxsackie viruses A24 and B3, and the three VPgs of FMDV. The areas of the VPg visible in the crystal structures of CVB3 and FMDV-VPg1 (illustrated in Figure 4) in complex with their polymerases are underlined.

Figure 2. Images from spectra used for assignment

A. section of the NOESY spectrum, showing NOE cross peaks from the aromatic (δ and ε) protons of the uridylylated Tyr3 side chain (TYU3) to the rest of the peptide. A hand assignment is shown for the more obvious peaks. The position of crosspeaks to the side chain amines of ASN8 and ASN12 are also indicated. B. Natural abundance ¹⁵N-HSQC spectrum, showing the assignments for the sidechains (SC) of Asn8, Asn12, and Q22 and the backbone amide assignments (found for all except G1 and A2). C. The well resolved natural abundance ¹³C-HMQC spectra was particularly useful for refining the assignment of the sidechain and UMP atoms (labels).

Figure 2. Images from spectra used for assignment

A. section of the NOESY spectrum, showing NOE cross peaks from the aromatic (δ and ε) protons of the uridylylated Tyr3 side chain (TYU3) to the rest of the peptide. A hand assignment is shown for the more obvious peaks. The position of crosspeaks to the side chain amines of ASN8 and ASN12 are also indicated. B. Natural abundance ¹⁵N-HSQC spectrum, showing the assignments for the sidechains (SC) of Asn8, Asn12, and Q22 and the backbone amide assignments (found for all except G1 and A2). C. The well resolved natural abundance ¹³C-HMQC spectra was particularly useful for refining the assignment of the sidechain and UMP atoms (labels).

Figure 2. Images from spectra used for assignment

A. section of the NOESY spectrum, showing NOE cross peaks from the aromatic (δ and ε) protons of the uridylylated Tyr3 side chain (TYU3) to the rest of the peptide. A hand assignment is shown for the more obvious peaks. The position of crosspeaks to the side chain amines of ASN8 and ASN12 are also indicated. B. Natural abundance ¹⁵N-HSQC spectrum, showing the assignments for the sidechains (SC) of Asn8, Asn12, and Q22 and the backbone amide assignments (found for all except G1 and A2). C. The well resolved natural abundance ¹³C-HMQC spectra was particularly useful for refining the assignment of the sidechain and UMP atoms (labels).

Figure 3. Structure of PV1-VPgpU

A) Bundle of the 10 top NMR structures for VPgpU; the TYU sidechain is green. B) Space filling depiction of the top structure, showing how two conserved positively charged residues residues Lys 9 and Lys 20 (violet)surround the TYU sidechain, which is colored according to atom type (Red:O, Blue: N, Black:C, Khaki: P, and gray:H), with atoms important for base-pairing and chain elongation labeled. C) Ribbon diagram showing distances < 4Å (purple lines) between sidechains from the rest of the VPg peptide to the TYU sidechain. The backbone is turquoise, the sidechains of Arg 17 and Lys 20 are blue, Lys 9 is orange and that of Gln22 is gold; the other sidechains are shown in light gray. The orientation of this figure is tilted slightly from that of PV1-VPgpU in Figure 3. D) overlay of the middle 9 residues of the solution structures of PV1-VPg (PDB file 2BBL; maroon), PV1-VPgpU (blue, current study), and the 9 residues visible in the structure of CVB3-VPg complexed with its polymerase[25] (gold).

Figure 4

Side by side comparison of PV1-VPg and PV1-VPgpU solution NMR structures with the partial crystal structures of CVB3 VPg (residues 7–15) and FMDV- VPgpU (residues 1–15).

Figure 5. 5-MeUTP does not inhibit VPg uridylylation in vitro when normal viral growth conditions are mimicked

Increasing amounts of cold 5-MeUTP, at the concentrations shown, were added to VPg uridylylation reactions with a constant amount (10 µM) of labeled UTP. The reaction was either specific, with the cre(2C) RNA (the hairpin loop that is the template in the viral RNA) with Mg²⁺ (left) or non-viral specific with a poly(A) template and Mn²⁺ (right). Activity is calculated relative to the amount of UTP incorporated in the absence of 5-MeUTP.

Figure 6. Docking places PV1-VPgpU into the active site of PV1-polymerase

but further from the nucleotide/ metal binding site (green residues in the polymerases) than FMDV-VPgpU is in a crystal structure with its polymerase. Left: A docked complex (Zdock score >50) of PV1-VPgpU (space filling, colored as in Figure 3B) with a crystal structure of PV1-Pol3D (PDB file 1RA6). Right: For comparison, a snapshot is shown of the position of the N-terminal 15 amino acids of FMDV-VPgpU (see Figure 3) in a crystal structure of its complex with the FMDV polymerase (PDB file 2F8E) [18]. Note the O4 of the Uridine base is in a different position relative to the active site residues of the polymerase in the docked complex, The polymerases are shown in ribbon format in a similar orientation, with the side chains of the three residues conserved in the active sites of all polymerases, that mediate metal ion binding (PV1: D328,D329 or for FMDV, D338,D339) and incoming base stabilization (Y326, for FMDV Y336)) colored green. Sidechains on the polymerase are labeled in the one letter code, those of VPgpU are three letter code. A dotted oval on each polymerase structure marks the area of the possible binding site for VPg on PV1-pol3D[56] (equivalent to that seen in the crystal structure of CVB3 complex[25]). Note that the corresponding area, also circled by an oval, on the FMDV polymerase (right) differs in sequence and secondary structure.