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Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

Published on: August 31, 2017

Viral attenuation by engineered protein fragmentation.

Daniel J Garry¹, Andrew D Ellington¹, Ian J Molineux²

¹Department of Molecular Biosciences, Center for Systems and Synthetic Biology, University of Texas, Austin, TX 78712, USA.

Virus Evolution

|June 27, 2018

Summary

This summary is machine-generated.

Protein fragmentation, a novel viral attenuation method, was tested by engineering bacteriophage T7 RNA polymerase into two self-assembling peptides. Adaptation over 100 generations significantly increased fitness, demonstrating feasibility but highlighting initial fitness as a poor predictor of long-term adaptation.

Keywords:

adaptation attenuation bacteriophage evolution genome engineering

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Area of Science:

Virology
Molecular Biology
Biotechnology

Background:

Protein fragmentation is a theoretical method for viral attenuation.
Engineering viral proteins into self-assembling peptides could reduce viral fitness.

Purpose of the Study:

To test the feasibility of protein fragmentation for viral attenuation.
To engineer bacteriophage T7 RNA polymerase into two self-assembling peptides.
To assess the long-term adaptation and fitness of the engineered virus.

Main Methods:

Bacteriophage T7 was genetically engineered to express its RNA polymerase as two separate peptides.
The engineered virus underwent 100 generations of adaptation via serial transfer.
Genomic analysis was performed to identify mutations associated with fitness recovery.

Main Results:

Initial fitness of the fragmented virus was significantly suppressed.
Adaptation led to substantial fitness recovery, though it remained below wild-type levels.
Fitness gains were associated with three substitutions in fragmented peptides and six genomic mutations.
The engineered fragmentation was maintained throughout adaptation.

Conclusions:

Gene fragmentation is a feasible strategy for viral attenuation.
Extended adaptation can significantly improve the fitness of fragmented viruses.
Initial fitness suppression may not accurately predict long-term adaptive potential.