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Reversion mutations in phosphoprotein P of a codon-pair-deoptimized human respiratory syncytial virus confer increased transcription, immunogenicity, and genetic stability without loss of attenuation
- Source :
- PLoS Pathogens, Vol 17, Iss 12, p e1010191 (2021), PLoS Pathogens
- Publication Year :
- 2021
- Publisher :
- Public Library of Science (PLoS), 2021.
-
Abstract
- Recoding viral genomes by introducing numerous synonymous nucleotide substitutions that create suboptimal codon pairs provides new live-attenuated vaccine candidates. Because recoding typically involves a large number of nucleotide substitutions, the risk of de-attenuation is presumed to be low. However, this has not been thoroughly studied. We previously generated human respiratory syncytial virus (RSV) in which the NS1, NS2, N, P, M and SH ORFs were codon-pair deoptimized (CPD) by 695 synonymous nucleotide changes (Min A virus). Min A exhibited a global reduction in transcription and protein synthesis, was restricted for replication in vitro and in vivo, and exhibited moderate temperature sensitivity. Here, we show that under selective pressure by serial passage at progressively increasing temperatures, Min A regained replication fitness and lost its temperature sensitivity. Whole-genome deep sequencing identified numerous missense mutations in several genes, in particular ones accumulating between codons 25 and 34 of the phosphoprotein (P), a polymerase cofactor and chaperone. When re-introduced into Min A, these P mutations restored viral transcription to wt level, resulting in increased protein expression and RNA replication. Molecular dynamic simulations suggested that these P mutations increased the flexibility of the N-terminal domain of P, which might facilitate its interaction with the nucleoprotein N, and increase the functional efficiency of the RSV transcription/replication complex. Finally, we evaluated the effect of the P mutations on Min A replication and immunogenicity in hamsters. Mutation P[F28V] paradoxically reduced Min A replication but not its immunogenicity. The further addition of one missense mutation each in M and L generated a version of Min A with increased genetic stability. Thus, this study provides further insight into the adaptability of large-scale recoded RNA viruses under selective pressure and identified an improved CPD RSV vaccine candidate.<br />Author summary Synonymous recoding of viral genomes by codon-pair deoptimization (CPD) generates live-attenuated vaccines presumed to be genetically stable due to the high number of nucleotide substitutions. However, their actual genetic stability under selective pressure was largely unknown. In a recoded human respiratory syncytial virus (RSV) mutant called Min A, six of 11 ORFs were CPD, reducing protein expression and inducing moderate temperature sensitivity and attenuation. When passaged in vitro under selective pressure, Min A lost its temperature-sensitive phenotype and regained fitness by the acquisition of numerous mutations, in particular missense mutations in the viral phosphoprotein (P), a polymerase cofactor and a chaperone for soluble nucleoprotein. These P mutations increased RSV gene transcription globally, thereby increasing RSV protein expression, RNA replication, and virus particle production. Thus, the P mutations increased the efficiency of the RSV transcription/replication complex, compensating for the reduced protein expression due to CPD. In addition, introduction of the P mutations into Min A generated a live-attenuated vaccine candidate with increased genetic stability. Surprisingly, this vaccine candidate exhibited increased attenuation and, paradoxically, exhibited increased immunogenicity per plaque-forming unit in hamsters. This study provides insights into the adaptability of large-scale recoded RNA viruses and identified an improved CPD RSV vaccine candidate.
- Subjects :
- Transcription, Genetic
Physiology
Cell Lines
Protein Expression
Biochemistry
Medical Conditions
Immune Physiology
Cricetinae
Chlorocebus aethiops
Medicine and Health Sciences
Biology (General)
Mammals
Vaccines
Immune System Proteins
Microbial Mutation
Eukaryota
Infectious Diseases
Vertebrates
Hamsters
Biological Cultures
Research Article
Missense Mutation
Infectious Disease Control
QH301-705.5
Immunology
Respiratory Syncytial Virus Infections
Research and Analysis Methods
Vaccines, Attenuated
Microbiology
Rodents
Virology
Genetics
Gene Expression and Vector Techniques
Respiratory Syncytial Virus Vaccines
Animals
Antigens
Molecular Biology Techniques
Vero Cells
Molecular Biology
Viral Structural Proteins
Molecular Biology Assays and Analysis Techniques
Mesocricetus
Organisms
Biology and Life Sciences
Proteins
Viral Vaccines
RC581-607
Phosphoproteins
Viral Replication
Respiratory Syncytial Virus, Human
Amniotes
Mutation
Parasitology
Immunologic diseases. Allergy
Zoology
Subjects
Details
- Language :
- English
- ISSN :
- 15537374 and 15537366
- Volume :
- 17
- Issue :
- 12
- Database :
- OpenAIRE
- Journal :
- PLoS Pathogens
- Accession number :
- edsair.doi.dedup.....50befa37870babd1f3980b6190f200af