Vaccine-derived Mutation in Motif D of Poliovirus RNA-dependent RNA Polymerase Lowers Nucleotide Incorporation Fidelity

Xinran Liu,Xiaorong Yang,Cheri A Lee,Ibrahim M Moustafa,Eric D Smidansky,David Lum,Jamie J Arnold,Craig E Cameron,David D Boehr

doi:10.1074/jbc.m113.484428

Xinran Liu, Xiaorong Yang + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m113.484428

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Abstract

All viral RNA-dependent RNA polymerases (RdRps) have a conserved structural element termed motif D. Studies of the RdRp from poliovirus (PV) have shown that a conformational change of motif D leads to efficient and faithful nucleotide addition by bringing Lys-359 into the active site where it serves as a general acid. The RdRp of the Sabin I vaccine strain has Thr-362 changed to Ile. Such a drastic change so close to Lys-359 might alter RdRp function and contribute in some way to the attenuated phenotype of Sabin type I. Here we present our characterization of the T362I RdRp. We find that the T362I RdRp exhibits a mutator phenotype in biochemical experiments in vitro. Using NMR, we show that this change in nucleotide incorporation fidelity correlates with a change in the structural dynamics of motif D. A recombinant PV expressing the T362I RdRp exhibits normal growth properties in cell culture but expresses a mutator phenotype in cells. For example, the T362I-containing PV is more sensitive to the mutagenic activity of ribavirin than wild-type PV. Interestingly, the T362I change was sufficient to cause a statistically significant reduction in viral virulence. Collectively, these studies suggest that residues of motif D can be targeted when changes in nucleotide incorporation fidelity are desired. Given the observation that fidelity mutants can serve as vaccine candidates, it may be possible to use engineering of motif D for this purpose.

Highlights

The motif D loop in poliovirus RNA-dependent RNA polymerase is important for catalysis and fidelity
T362I substitution in PV RNA-dependent RNA polymerases (RdRps) induces a decrease in polymerase fidelity (Figs. 3 and 4) by making the pre-chemistry conformational change more efficient (Fig. 5), likely through disrupting interactions in motif D and other regions of the active-site (Fig. 6), and PV strains expressing this variant have reduced virulence in mice (Fig. 7)
We have proposed that motif D in RdRps and reverse transcriptases play an analogous role to helix O/P in family A/B of DNA polymerases [30]; both regions contain a conserved Lys that acts as a general acid in phosphodiester bond formation [29], and both regions undergo fidelity-governing conformational changes (30, 64 –70)

Summary

Background

The motif D loop in poliovirus RNA-dependent RNA polymerase is important for catalysis and fidelity. Using NMR, we show that this change in nucleotide incorporation fidelity correlates with a change in the structural dynamics of motif D. The T362I change was sufficient to cause a statistically significant reduction in viral virulence These studies suggest that residues of motif D can be targeted when changes in nucleotide incorporation fidelity are desired. A strategy to rationally design live, attenuated vaccine strains has been proposed based on modifying RdRps to change their nucleotide incorporation fidelity [12, 13]; viral strains encoding RdRps with altered fidelity (either higher or lower fidelity) have been shown to lead to virus incapable of causing disease but capable of acting as vaccine strains to protect mice from a lethal challenge from wild-type virus [12, 13].

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

36 Ϯ 2 33 Ϯ 2

DISCUSSION