Directed Evolution of an Error‐Prone T7 DNA Polymerase that Attenuates Viral Replication

Söte, Sandra; Kleine, Stefan; Schlicke, Marina; Brakmann, Susanne

doi:10.1002/cbic.201000799

Cited by 7 publications

(3 citation statements)

References 47 publications

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“…Our analysis did not account for mutational errors resulting from the in vitro transcription step. However, the T7 DNA-dependent RNA polymerase has been reported to have an error rate of only about 6 ϫ 10 Ϫ6 mutation/nucleotide (37), which is 10-to 1,000-fold lower than the estimated rate of mutation in ss(ϩ) RNA and thus should not have influenced our analysis. PEDEL-AA predicts high library diversity at the protein level and suggests that the Ep-PCR libraries should be highly useful at the functional level.…”

Section: Discussionmentioning

confidence: 74%

Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1

Agarwal

Baccam²,

Aggarwal

et al. 2018

J Virol

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Many RNA viruses exist as an ensemble of genetically diverse, replicating populations known as a mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds with various levels of genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70% of open reading frame 1 () or of the full genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency on the order of 10 per nucleotide copied and the existence of marked genetic diversity, with a high normalized Shannon entropy value. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of various sizes and compositions on phenotypic characteristics, such as the replication level of (+)-RNA progeny, the amounts of double-stranded RNA (a surrogate for the rate of viral replication) and ORF1 protein, and the expression of interferon-stimulated genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help to provide further insights into virus evolution and prediction of emerging viral diseases. Several biological or practical limitations currently prevent the study of phenotypic behavior of a mutant cloud We developed a simple and rapid method for synthesizing mutant clouds of hepatitis E virus (HEV), a single-stranded (+)-RNA [ss(+) RNA] virus, with various and controllable levels of genetic diversity, which could then be used in a cell culture system to study the effects of cloud size and composition on viral phenotype. In a cross-sectional analysis, we demonstrated that a particular mutant cloud which had an extremely high genetic diversity had a replication rate exceeding that of wild-type HEV. This method should thus provide a useful model for understanding the phenotypic behavior of ss(+) RNA viruses.

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Section: Discussionmentioning

confidence: 74%

Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1

Agarwal

Baccam²,

Aggarwal

et al. 2018

J Virol

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“…Because Pol I is required for the replication of DNA immediately downstream of a ColE1 origin of replication, genes included within this region may be used to provide specific selection pressures. For example, Brakmann and co‐workers evolved a mutagenic variant of T7 DNAP by selecting for mutational reversion of a β‐lactamase gene and growth in the presence of ampicillin [56].…”

Section: Screening and Selection Strategiesmentioning

confidence: 99%

Directed polymerase evolution

Chen

Romesberg

2013

FEBS Letters

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Polymerases evolved in nature to synthesize DNA and RNA, and they underlie the storage and flow of genetic information in all cells. The availability of these enzymes for use at the bench has driven a revolution in biotechnology and medicinal research; however, polymerases did not evolve to function efficiently under the conditions required for some applications and their high substrate fidelity precludes their use for most applications that involve modified substrates. To circumvent these limitations, researchers have turned to directed evolution to tailor the properties and/or substrate repertoire of polymerases for different applications, and several systems have been developed for this purpose. These systems draw on different methods of creating a pool of randomly mutated polymerases and are differentiated by the process used to isolate the most fit members. A variety of polymerases have been evolved, providing new or improved functionality, as well as interesting new insight into the factors governing activity.

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“…Besides, two other studies focused as well on protein engineering, respectively for a better catalytic activity [147] and a stronger binding ability [148] . Interestingly, coupled with β-lactamase assay, the platform was also reversely employed to guide the directed evolution of active polymerases with reduced fidelity [149] , [150] . As for the yeast version, it is more like an orthogonal module in synthetic biology, and was proposed by many to help build synthetic circuits [151] , [152] and sustain biological containment [153] , [154] .…”

Section: Existing Targeted Mutagenesis Methodsmentioning

confidence: 99%

Targeted mutagenesis: A sniper-like diversity generator in microbial engineering

Zheng

Xing

Zhang

2017

Synthetic and Systems Biotechnology

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Mutations, serving as the raw materials of evolution, have been extensively utilized to increase the chances of engineering molecules or microbes with tailor-made functions. Global and targeted mutagenesis are two main methods of obtaining various mutations, distinguished by the range of action they can cover. While the former one stresses the mining of novel genetic loci within the whole genomic background, targeted mutagenesis performs in a more straightforward manner, bringing evolutionary escape and error catastrophe under control. In this review, we classify the existing techniques of targeted mutagenesis into two categories in terms of whether the diversity is generated in vitro or in vivo, and briefly introduce the mechanisms and applications of them separately. The inherent connections and development trends of the two classes are also discussed to provide an insight into the next generation evolution research.

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Directed Evolution of an Error‐Prone T7 DNA Polymerase that Attenuates Viral Replication

Cited by 7 publications

References 47 publications

Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1

Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1

Directed polymerase evolution

Targeted mutagenesis: A sniper-like diversity generator in microbial engineering

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