Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles.

Edward I Patterson,Saravanan Thangamani,Naomi L Forrester,Kamil Khanipov,Daniele M Swetnam,Samantha Walsdorf,Tiffany F Kautz,Yuriy Fofanov

doi:10.3390/v12050546

Abstract

Mutations are incorporated into the genomes of RNA viruses at an optimal frequency and altering this precise frequency has been proposed as a strategy to create live-attenuated vaccines. However, determining the effect of specific mutations that alter fidelity has been difficult because of the rapid selection of the virus population during replication. By deleting residues of the structural polyprotein PE2 cleavage site, E3Δ56-59, in Venezuelan equine encephalitis virus (VEEV) TC-83 vaccine strain, non-infectious virus particles were used to assess the effect of single mutations on mutation frequency without the interference of selection that results from multiple replication cycles. Next-generation sequencing analysis revealed a significantly lower frequency of transversion mutations and overall mutation frequency for the fidelity mutants compared to VEEV TC-83 E3Δ56-59. We demonstrate that deletion of the PE2 cleavage site halts virus infection while making the virus particles available for downstream sequencing. The conservation of the site will allow the evaluation of suspected fidelity mutants across alphaviruses of medical importance.

Highlights

IntroductionViruses 2020, 12, x FOR PEER REVIEW mutation rate is high enough to be beneficial, while the number of deleterious mutations remain below belowthat levels thatotherwise would otherwise error catastrophe, stateexcessive where excessive mutations levels would result inresult error in catastrophe, the statethe where mutations render render proteins non-functional and can potentially lead to viral extinction
To determine whether the rescued viruses were infectious, a Cytopathic effect (CPE) assay was performed on media collected at 24 h and 48 h post-electroporation from unmutated Venezuelan equine encephalitis virus (VEEV) TC-83 control, TC-83 E3∆56-59, and TC-83 E1-G91D
Wells inoculated with harvested media from TC-83 E3∆56-59 and TC-83 E1-G91D electroporations were negative for CPE, while the TC-83 control wells were positive for CPE

Summary

Introduction

Viruses 2020, 12, x FOR PEER REVIEW mutation rate is high enough to be beneficial, while the number of deleterious mutations remain below belowthat levels thatotherwise would otherwise error catastrophe, stateexcessive where excessive mutations levels would result inresult error in catastrophe, the statethe where mutations render render proteins non-functional and can potentially lead to viral extinction. If this mutation rate is proteins non-functional and can potentially lead to viral extinction. If this mutation rate is altered, altered, even slightly, the resulting virus population commonly exhibits attenuation, especially in even slightly, the resulting virus population commonly exhibits attenuation, especially in vivo [3,4,5,6,7,8,9,10,11,12]

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