Abstract
The replication machinery of most RNA viruses lacks proofreading mechanisms. As a result, RNA virus populations harbor a large amount of genetic diversity that confers them the ability to rapidly adapt to changes in their environment. In this work, we investigate whether further increasing the initial population diversity of a model RNA virus can improve adaptation to a single selection pressure, thermal inactivation. For this, we experimentally increased the diversity of coxsackievirus B3 (CVB3) populations across the capsid region. We then compared the ability of these high diversity CVB3 populations to achieve resistance to thermal inactivation relative to standard CVB3 populations in an experimental evolution setting. We find that viral populations with high diversity are better able to achieve resistance to thermal inactivation at both the temperature employed during experimental evolution as well as at a more extreme temperature. Moreover, we identify mutations in the CVB3 capsid that confer resistance to thermal inactivation, finding significant mutational epistasis. Our results indicate that even naturally diverse RNA virus populations can benefit from experimental augmentation of population diversity for optimal adaptation and support the use of such viral populations in directed evolution efforts that aim to select viruses with desired characteristics.
Highlights
The replication machinery of most RNA viruses lacks proofreading mechanisms
A high rate of background mutations was observed for single mutations within codons compared to a non-mutagenized library (WT Lib; Fig. 1B; Table S3), while two or three mutations per codon showed > 400-fold higher rates in the mutagenized libraries compared to the non-mutagenized library (Fig. 1B; Table S3)
Previous studies using high-fidelity polymerase mutants have shown that RNA virus populations with reduced diversity are less able to adapt to new environments compared to standard virus populations[10,11,30]
Summary
The replication machinery of most RNA viruses lacks proofreading mechanisms. As a result, RNA virus populations harbor a large amount of genetic diversity that confers them the ability to rapidly adapt to changes in their environment. In such directed evolution experiments, virus populations are grown under conditions that favor either the emergence or further optimization of the desired phenotype This process has been used to obtain live attenuated vaccines for numerous viruses by repeated growth at suboptimal conditions[3], improving in vivo models by serial infection of the desired h ost[4,5,6,7,8,9], isolating RNA viruses with high-fidelity polymerases by growth under condition of increased mutational load[10,11], selection of viruses with improved oncolytic p roperties[12,13,14], or isolation of viruses and virus-like particles of increased stability[15,16,17]. We apply a codon-level mutagenesis protocol to the entire capsid region of the human picornavirus coxsackievirus B3 (CVB3) and generate viral populations with increased diversity across the capsid protein Using these CVB3 populations, we examine if RNA viruses can further benefit from an initial increase in diversity to adapt to a single selection pressure, thermal inactivation of the capsid. Our results indicate that even RNA viruses with extreme mutation rates can further benefit from augmentation of population diversity for adaptation, and support the use of such viral populations in directed evolution experiments
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