Allosteric Effects of Ligands and Mutations on Poliovirus RNA-Dependent RNA Polymerase

Abstract

Protein priming of viral RNA synthesis plays an essential role in the replication of picornavirus RNA. Both poliovirus and coxsackievirus encode a small polypeptide, VPg, which serves as a primer for addition of the first nucleotide during synthesis of both positive and negative strands. This study examined the effects on the VPg uridylylation reaction of the RNA template sequence, the origin of VPg (coxsackievirus or poliovirus), the origin of 3D polymerase (coxsackievirus or poliovirus), the presence and origin of interacting protein 3CD, and the introduction of mutations at specific regions in the poliovirus 3D polymerase. Substantial effects associated with VPg origin were traced to differences in VPg-polymerase interactions. The effects of 3CD proteins and mutations at polymerase-polymerase intermolecular Interface I were most consistent with allosteric effects on the catalytic 3D polymerase molecule. In conclusion, the efficiency and specificity of VPg uridylylation by picornavirus polymerases is greatly influenced by allosteric effects of ligand binding that are likely to be relevant during the viral replicative cycle.Picornaviruses have a single copy, positive-strand RNA genome with a small peptide, VPg (3B), covalently attached to the 5Ј-terminal nucleotide and a poly(A) segment at the 3Ј end. RNA replication occurs in the cytoplasm of infected cells, proceeding through negative-strand intermediates which, in turn, serve as templates for production of progeny positive strands. A long-standing question has been how both positiveand negative-strand syntheses are initiated. Characterization of a reaction catalyzed by poliovirus 3D polymerase, in which the tyrosine of the VPg peptide was trans-esterified (uridylylated) in the presence of a poly(A) template to form VPg-pU (pU), provided insight into the mechanism of strand initiation (34): given the ability of poliovirus 3D polymerase to uridylylate VPg, the peptide-nucleotide conjugate could serve as the protein primer for progeny RNA strand synthesis. VPg must bind directly to poliovirus polymerase, because it can serve as an enzymatic substrate; furthermore, VPg-polymerase interactions have been observed in two-hybrid experiments (42). While the binding site on the poliovirus 3D polymerase for the VPg substrate of the uridylylation reaction has not yet been characterized, the binding site for its likely proteolytic precursor, 3AB, has been identified as a distinct site on the back of the palm of the polymerase molecule via extensive alaninescanning mutagenesis (22).Although VPg uridylylation could provide a protein primer for use in either positive-or negative-strand synthesis, this reaction is not sufficient to describe the mechanism of initiation for viral RNA synthesis in infected cells. For example, the use of a poly(A) template for VPg uridylylation does not provide specificity for a particular virus. This specificity could be provided if an RNA sequence or structure within the picornavirus genome were the authentic RNA template for VPg uridylylation...

The Crystal Structure of Coxsackievirus B3 RNA-Dependent RNA Polymerase in Complex with Its Protein Primer VPg Confirms the Existence of a Second VPg Binding Site on Picornaviridae Polymerases

“…1C). This corresponds to observations reported by others where VPg uridylation by PV and CVB3 RdRps in vitro needed to be activated either by Mn 2ϩ or, when Mg 2ϩ is used, by the presence of 3CD pro or 3C Pro (4,(45)(46)(47)(48)54). Another product, migrating slower in Tris-Tricine PAGE, (Fig.…”

“…Only in the presence of Mn 2ϩ , poly(rA), and VPg, did CVB3 3D pol generate two bands. The faster-migrating double-band can be attributed to VPg-pU(pU) (4,48,53). The slower-migrating band might correspond to VPg-poly(U) produced by primer elongation on poly(rA), 3D…”

“…pol -pU(pU), which has been shown to be generated by intra-or intermolecular uridylation of PV 3D pol (53), or to poly(rA)-(pU) n generated by a terminal transferase activity of 3D pol (4,41 on May 11, 2018 by guest http://jvi.asm.org/ molecular replacement using the structure of PV RdRp as a search model. The complex structure with VPg (resolution, 2.5 Å) was obtained using crystals grown in a mix of VPg, an oligo(rA) 6 template, 3ЈdUTP, and Mg 2ϩ .…”

“…The CVB3 3D pol /VPg complex was obtained by preparing a mix of 220 M CVB3 3D pol (in 20 mM Tris [pH 9.0], 300 mM NaCl, 15% glycerol, 0.5 mM Tris(2-carboxyethyl) phosphine), 440 M VPg (in 10 mM Tris [pH 7.5], 25 mM NaCl, and 10% glycerol), 1 mM oligoA 6 , 2.5 mM 3ЈdUTP, and 10 mM MgCl 2 . The mix was incubated for 24 h on ice prior to crystallization using two parts 2.0 M (NH 4 ) 2 SO 4 -100 mM CAPS (pH 10)-200 mM Li 2 SO 4 and one part 1.3 to 1.8 M NH 4 Cl-100 mM sodium acetate (pH 4.6 to 5.2) by volume (27). Tetragonal crystals (P4 3 2 1 2, a ϭ b ϭ 74.38 Å, c ϭ 285.81 Å) appeared 3 weeks after mixing the sample solution with the well buffer at a ratio of 4.3 to 3.7 (vol/vol).…”

Membrane Topography of the Hydrophobic Anchor Sequence of Poliovirus 3A and 3AB Proteins and the Functional Effect of 3A/3AB Membrane Association upon RNA Replication

“…Whether the TM and/or non-TM forms of 3A derived from membrane-bound 3AB, have a subsequent function in replication remains to be determined. The 3D pol /VPg complex then binds to 3CD pro by means of an interaction between 3D pol and the 3C pro domain of 3CD pro (42,66). Using the RNA binding activity of 3CD pro the complex is attached to the cre(2C) RNA where the uridylylation of VPg takes place (33,67).…”