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

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. Author summarySynonymous 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.