Low-Fidelity Polymerases of Alphaviruses Recombine at Higher Rates To Overproduce Defective Interfering Particles.

Enzo Z Poirier,Peter Jan Hooikaas,Gonzalo Moratorio,Bryan C Mounce,Kathryn Rozen-Gagnon,Marco Vignuzzi,Kenneth A Stapleford,T S Dermody

doi:10.1128/jvi.02921-15

Abstract

ABSTRACTLow-fidelity RNA-dependent RNA polymerases for many RNA virus mutators have been shown to confer attenuated phenotypes, presumably due to increased mutation rates. Additionally, for many RNA viruses, replication to high titers results in the production of defective interfering particles (DIs) that also attenuate infection. We hypothesized that fidelity, recombination, and DI production are tightly linked. We show that a Sindbis virus mutator replicating at a high multiplicity of infection manifests an earlier and greater accumulation of DIs than its wild-type counterpart. The isolated DIs interfere with the replication of full-length virus in a dose-dependent manner. Importantly, the ability of the mutator virus to overproduce DIs could be linked to an increased recombination frequency. These data confirm that RNA-dependent RNA polymerase fidelity and recombination are inversely correlated for this mutator. Our findings suggest that defective interference resulting from higher recombination rates may be more detrimental to RNA virus mutators than the increase in mutational burden.IMPORTANCE Replication, adaptation, and evolution of RNA viruses rely in large part on their low-fidelity RNA-dependent RNA polymerase. Viruses artificially modified in their polymerases to decrease fidelity (mutator viruses) are attenuated in vivo, demonstrating the important role of fidelity in viral fitness. However, attenuation was attributed solely to the modification of the viral mutation rate and the accumulation of detrimental point mutations. In this work, we described an additional phenotype of mutator viruses: an increased recombination rate leading to defective interfering particle (DI) overproduction. Because DIs are known for their inhibitory effect on viral replication, our work suggests that fidelity variants may be attenuated in vivo via several mechanisms. This has important implications in the development of fidelity variants as live attenuated vaccine strains.

Highlights

Low-fidelity RNA-dependent RNA polymerases for many RNA virus mutators have been shown to confer attenuated phenotypes, presumably due to increased mutation rates
We described an additional phenotype of mutator viruses: an increased recombination rate leading to defective interfering particle (DI) overproduction
Our findings demonstrate that along with increased error rate, higher recombination rates are a general characteristic of the RNA-dependent RNA polymerase (RdRp) mutator

Summary

Introduction

Low-fidelity RNA-dependent RNA polymerases for many RNA virus mutators have been shown to confer attenuated phenotypes, presumably due to increased mutation rates. This past decade, the engineering of RNA viruses of various families with altered mutation rates has shown that single substitutions can significantly alter polymerase activity and fidelity Among these variants, RdRp mutators present point mutations in the viral polymerase that lead to increased mutation rates compared to those of the wild-type (WT) virus. While many determinants that alter RdRp fidelity have been identified, the enzymatic mechanisms or dynamics by which this occurs are not well elucidated, and how decreasing fidelity may affect other polymerase activities is unclear [9, 10] Another curious feature of RNA virus replication is the production of truncated genomes and defective interfering particles (DIs).

Methods

Results

Conclusion