Dataset Information

Viral attenuation by engineered protein fragmentation.

ABSTRACT: A possible but untested method of viral attenuation is protein fragmentation, engineering wild-type proteins as two or more peptides that self-assemble after translation. Here, the bacteriophage T7 was engineered to encode its essential RNA polymerase as two peptides. Initial fitness was profoundly suppressed. Subjecting the engineered virus to over 100 generations of adaptation by serial transfer resulted in a large fitness increase, still remaining below that of evolved wild-type. The fitness increase was accompanied by three substitutions in the fragmented peptides as well as six mutations in other parts of the genome, but the fragmentation was retained. This study thereby demonstrates the feasibility of using gene fragmentation as a possibly permanent method of attenuation, but the initial fitness of the engineered genome may be a poor measure of its fitness on extended adaptation.

SUBMITTER: Garry DJ

PROVIDER: S-EPMC6009699 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Viral attenuation by engineered protein fragmentation.

Garry Daniel J DJ Ellington Andrew D AD Molineux Ian J IJ Bull James J JJ

Virus evolution 20180101 1

A possible but untested method of viral attenuation is protein fragmentation, engineering wild-type proteins as two or more peptides that self-assemble after translation. Here, the bacteriophage T7 was engineered to encode its essential RNA polymerase as two peptides. Initial fitness was profoundly suppressed. Subjecting the engineered virus to over 100 generations of adaptation by serial transfer resulted in a large fitness increase, still remaining below that of evolved wild-type. The fitness ...[more]

PMID: 29942657

Similar Datasets

Project description:Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3Dpol) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allow FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3Dpol in combination with previously reported results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon. Using biochemical and genetic tools, we show that the replacement of tryptophan 237 with phenylalanine imparts higher fidelity, but replacements with isoleucine and leucine resulted in lower-fidelity phenotypes. Viruses containing these W237 substitutions show in vitro growth kinetics and plaque morphologies similar to those of the wild-type (WT) A24 Cruzeiro strain in BHK cells, and both high- and low-fidelity variants retained fitness during coinfection with the wild-type virus. The higher-fidelity W237F (W237FHF) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237ILF) and W237LLF mutant viruses exhibited lower ribavirin resistance. Interestingly, the variant viruses showed heterogeneous and slightly delayed growth kinetics in primary porcine kidney cells, and they were significantly attenuated in mouse infection experiments. These data demonstrate, for a single virus, that either increased or decreased RdRp fidelity attenuates virus growth in animals, which is a desirable feature for the development of safer and genetically more stable vaccine candidates.IMPORTANCE Foot-and-mouth disease (FMD) is the most devastating disease affecting livestock worldwide. Here, using structural and biochemical analyses, we have identified FMDV 3Dpol mutations that affect polymerase fidelity. Recombinant FMDVs containing substitutions at 3Dpol tryptophan residue 237 were genetically stable and displayed plaque phenotypes and growth kinetics similar to those of the wild-type virus in cell culture. We further demonstrate that viruses harboring either a W237FHF substitution or W237ILF and W237LLF mutations were highly attenuated in animals. Our study shows that obtaining 3Dpol fidelity variants by protein engineering based on polymerase structure and function could be exploited for the development of attenuated FMDV vaccine candidates that are safer and more stable than strains obtained by selective pressure via mutagenic nucleotides or adaptation approaches.

Project description: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.

Dataset Information

Viral attenuation by engineered protein fragmentation.

Publications

Viral attenuation by engineered protein fragmentation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets