Combinatorial Approaches to Viral Attenuation

ABSTRACT Attenuated viruses have numerous applications, in particular in the context of live viral vaccines. However, purposefully designing attenuated viruses remains challenging, in particular if the attenuation is meant to be resistant to rapid evolutionary recovery. Here we develop and analyze a...

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Autores principales:	Matthew L. Paff, Benjamin R. Jack, Bartram L. Smith, James J. Bull, Claus O. Wilke
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	bacteriophages codon deoptimization promoter knockout viral attenuation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/291200245dde4ac385767d4c89da75c5
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spelling	oai:doaj.org-article:291200245dde4ac385767d4c89da75c52021-12-02T19:45:30ZCombinatorial Approaches to Viral Attenuation10.1128/mSystems.00046-182379-5077https://doaj.org/article/291200245dde4ac385767d4c89da75c52018-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00046-18https://doaj.org/toc/2379-5077ABSTRACT Attenuated viruses have numerous applications, in particular in the context of live viral vaccines. However, purposefully designing attenuated viruses remains challenging, in particular if the attenuation is meant to be resistant to rapid evolutionary recovery. Here we develop and analyze a new attenuation method, promoter ablation, using an established viral model, bacteriophage T7. Ablation of promoters of the two most highly expressed T7 proteins (scaffold and capsid) led to major reductions in transcript abundance of the affected genes, with the effect of the double knockout approximately additive of the effects of single knockouts. Fitness reduction was moderate and also approximately additive; fitness recovery on extended adaptation was partial and did not restore the promoters. The fitness effect of promoter knockouts combined with a previously tested codon deoptimization of the capsid gene was less than additive, as anticipated from their competing mechanisms of action. In one design, the engineering created an unintended consequence that led to further attenuation, the effect of which was studied and understood in hindsight. Overall, the mechanisms and effects of genome engineering on attenuation behaved in a predictable manner. Therefore, this work suggests that the rational design of viral attenuation methods is becoming feasible. IMPORTANCE Live viral vaccines rely on attenuated viruses that can successfully infect their host but have reduced fitness or virulence. Such attenuated viruses were originally developed through trial and error, typically by adaptation of the wild-type virus to novel conditions. That method was haphazard, with no way of controlling the degree of attenuation or the number of attenuating mutations or preventing evolutionary reversion. Synthetic biology now enables rational design and engineering of viral attenuation, but rational design must be informed by biological principles to achieve stable, quantitative attenuation. This work shows that in a model system for viral attenuation, bacteriophage T7, attenuation can be obtained from rational design principles, and multiple different attenuation approaches can be combined for enhanced overall effect.Matthew L. PaffBenjamin R. JackBartram L. SmithJames J. BullClaus O. WilkeAmerican Society for Microbiologyarticlebacteriophagescodon deoptimizationpromoter knockoutviral attenuationMicrobiologyQR1-502ENmSystems, Vol 3, Iss 4 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	bacteriophages codon deoptimization promoter knockout viral attenuation Microbiology QR1-502
spellingShingle	bacteriophages codon deoptimization promoter knockout viral attenuation Microbiology QR1-502 Matthew L. Paff Benjamin R. Jack Bartram L. Smith James J. Bull Claus O. Wilke Combinatorial Approaches to Viral Attenuation
description	ABSTRACT Attenuated viruses have numerous applications, in particular in the context of live viral vaccines. However, purposefully designing attenuated viruses remains challenging, in particular if the attenuation is meant to be resistant to rapid evolutionary recovery. Here we develop and analyze a new attenuation method, promoter ablation, using an established viral model, bacteriophage T7. Ablation of promoters of the two most highly expressed T7 proteins (scaffold and capsid) led to major reductions in transcript abundance of the affected genes, with the effect of the double knockout approximately additive of the effects of single knockouts. Fitness reduction was moderate and also approximately additive; fitness recovery on extended adaptation was partial and did not restore the promoters. The fitness effect of promoter knockouts combined with a previously tested codon deoptimization of the capsid gene was less than additive, as anticipated from their competing mechanisms of action. In one design, the engineering created an unintended consequence that led to further attenuation, the effect of which was studied and understood in hindsight. Overall, the mechanisms and effects of genome engineering on attenuation behaved in a predictable manner. Therefore, this work suggests that the rational design of viral attenuation methods is becoming feasible. IMPORTANCE Live viral vaccines rely on attenuated viruses that can successfully infect their host but have reduced fitness or virulence. Such attenuated viruses were originally developed through trial and error, typically by adaptation of the wild-type virus to novel conditions. That method was haphazard, with no way of controlling the degree of attenuation or the number of attenuating mutations or preventing evolutionary reversion. Synthetic biology now enables rational design and engineering of viral attenuation, but rational design must be informed by biological principles to achieve stable, quantitative attenuation. This work shows that in a model system for viral attenuation, bacteriophage T7, attenuation can be obtained from rational design principles, and multiple different attenuation approaches can be combined for enhanced overall effect.
format	article
author	Matthew L. Paff Benjamin R. Jack Bartram L. Smith James J. Bull Claus O. Wilke
author_facet	Matthew L. Paff Benjamin R. Jack Bartram L. Smith James J. Bull Claus O. Wilke
author_sort	Matthew L. Paff
title	Combinatorial Approaches to Viral Attenuation
title_short	Combinatorial Approaches to Viral Attenuation
title_full	Combinatorial Approaches to Viral Attenuation
title_fullStr	Combinatorial Approaches to Viral Attenuation
title_full_unstemmed	Combinatorial Approaches to Viral Attenuation
title_sort	combinatorial approaches to viral attenuation
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/291200245dde4ac385767d4c89da75c5
work_keys_str_mv	AT matthewlpaff combinatorialapproachestoviralattenuation AT benjaminrjack combinatorialapproachestoviralattenuation AT bartramlsmith combinatorialapproachestoviralattenuation AT jamesjbull combinatorialapproachestoviralattenuation AT clausowilke combinatorialapproachestoviralattenuation
_version_	1718376015089106944

Combinatorial Approaches to Viral Attenuation

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