Picornavirus Genome Replication

Abstract

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.

Picornavirus Genome Replication

“…1B. This model is consistent with much of what is known about this reaction (2-5, 7, 8, 11-19), but the details have emerged from studies performed in vitro on minimal templates and minimal protein domains (2)(3)(4)(5). Briefly, two molecules of 3C(D) bind to oriI (step 1) (3).…”

“…Importantly, this model explains and predicts biological phenotypes (2,4,5,20). However, VPg uridylylation occurs 10 -50-fold slower in vitro than necessary to support the rate constant (ϳ0.1/s) for initiation calculated from biological data (see Ref.…”

“…Our laboratory has been quite interested in defining the molecular details of VPg attachment to the 5Ј end of picornaviral RNAs (2)(3)(4)(5). This reaction is thought to occur in two independent half-reactions catalyzed by the viral RNA-dependent RNA polymerase, 3Dpol.…”

Long-Range Communication between Different Functional Sites in the Picornaviral 3C Protein

“…Perhaps even more remarkable was the finding that the chemical shift positions of these resonances depended on whether SL RNA or CA peptide was added first (Figure 3B). Many of the residues associated with multiple resonances in the ternary complexes (Figure 3C) are near the 3C dimer interface as observed by crystallographic studies (Mosimann et al, 1997) and/or near interfaces proposed to be important for interacting with other PV proteins (Shen et al, 2008). …”

“…Other residues are important in forming higher order complexes with other viral proteins. For example, a structural model involving a 3C dimer and a 3D monomer binding to oriI RNA has been proposed to be important for initiation of RNA synthesis (Shen et al, 2008). Many of the 3C residues that form intersubunit interactions within this complex are conformationally dynamic on the millisecond timescale, as shown either by the R 2 relaxation dispersion experiments (Figure 4) or by the presence of multiple resonances in the ternary complexes (Figure 3), including residues important for 3C-3D interactions such as Phe25, Lys108 and Asn165 and residues important for 3C-3C interactions such as Lys60, Ala61 and Asn69.…”