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Evolutionary recovery of a recombinant viral genome.

ABSTRACT: It is well appreciated that the evolutionary divergence of genes and genomes from a common ancestor ultimately leads to incompatibilities if those genomes are hybridized. Far less is known about the ability and nature of compensatory evolution to yield the recovery of function in hybrid genomes. Here the major capsid gene of the bacteriophage T7 (40-kb dsDNA) was replaced with the homologous gene of either T3 or K11, each 22% different at the protein level from the T7 homolog. Initial fitness was moderately impaired for the T3 exchange, but the K11 exchange was not viable without a compensatory change in the T7 scaffolding protein. Subsequent adaptation of the transgenic phages led to nearly complete fitness recoveries. Compensatory changes were few, mostly in the transgene and its main interacting partner, the scaffolding protein gene. The large magnitude of fitness recovery with relatively few mutations suggests that the fitness costs of hybridizations and horizontal gene exchanges between moderately diverged genomes can potentially be short-lived through compensatory evolution.

SUBMITTER: Springman R

PROVIDER: S-EPMC3385988 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Evolutionary recovery of a recombinant viral genome.

Springman Rachael R Kapadia-Desai Devanshi S DS Molineux Ian J IJ Bull James J JJ

G3 (Bethesda, Md.) 20120701 7

It is well appreciated that the evolutionary divergence of genes and genomes from a common ancestor ultimately leads to incompatibilities if those genomes are hybridized. Far less is known about the ability and nature of compensatory evolution to yield the recovery of function in hybrid genomes. Here the major capsid gene of the bacteriophage T7 (40-kb dsDNA) was replaced with the homologous gene of either T3 or K11, each 22% different at the protein level from the T7 homolog. Initial fitness wa ...[more]

PMID: 22870405

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Project description:Precise, reproducible characterization of AAV is critical for comparing preclinical results between laboratories and determining a safe and effective clinical dose for gene therapy applications. In this study, we systematically evaluated numerous parameters to produce a simple and robust ddPCR protocol for AAV characterization. The protocol uses a low ionic strength buffer containing Pluronic-F68 and polyadenylic acid to dilute the AAV into the ddPCR concentration range and a 10-minute thermal capsid lysis prior to assembling ddPCR reactions containing MspI. A critical finding is that the buffer composition affected the ITR concentration of AAV but not the ITR concentration of a double stranded plasmid, which has implications when using a theoretical, stoichiometric conversion factor to obtain the titer based on the ITR concentration. Using this protocol, a more comprehensive analysis of an AAV vector formulation was demonstrated with multiple ddPCR assays distributed throughout the AAV vector genome. These assays amplify the ITR, regulatory elements, and eGFP transgene to provide a more confident estimate of the vector genome concentration and a high-resolution characterization of the vector genome identity. Additionally, we compared two methods of genome integrity analysis for three control sample types at eight different concentrations for each sample. The genome integrity was independent of sample concentration and the expected values were obtained when integrity was determined based on the excess number of positive droplets relative to the number of double positive droplets expected by chance co-encapsulation of two DNA targets. The genome integrity was highly variable and produced unexpected values when the double positive droplet percentage was used to calculate the genome integrity. A protocol using a one-minute thermal capsid lysis prior to assembling ddPCR reactions lacking a restriction enzyme used the non-ITR assays in a duplex ddPCR milepost experiment to determine the genome integrity using linkage analysis.

Dataset Information

Evolutionary recovery of a recombinant viral genome.

Publications

Evolutionary recovery of a recombinant viral genome.

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