Rescue of codon-pair deoptimized respiratory syncytial virus by the emergence of genomes with very large internal deletions that complemented replication

Proc Natl Acad Sci U S A. 2021 Mar 30;118(13):e2020969118. doi: 10.1073/pnas.2020969118.


Recoding viral genomes by introducing numerous synonymous but suboptimal codon pairs-called codon-pair deoptimization (CPD)-provides new types of live-attenuated vaccine candidates. The large number of nucleotide changes resulting from CPD should provide genetic stability to the attenuating phenotype, but this has not been rigorously tested. Human respiratory syncytial virus in which the G and F surface glycoprotein ORFs were CPD (called Min B) was temperature-sensitive and highly restricted in vitro. When subjected to selective pressure by serial passage at increasing temperatures, Min B substantially regained expression of F and replication fitness. Whole-genome deep sequencing showed many point mutations scattered across the genome, including one combination of six linked point mutations. However, their reintroduction into Min B provided minimal rescue. Further analysis revealed viral genomes bearing very large internal deletions (LD genomes) that accumulated after only a few passages. The deletions relocated the CPD F gene to the first or second promoter-proximal gene position. LD genomes amplified de novo in Min B-infected cells were encapsidated, expressed high levels of F, and complemented Min B replication in trans This study provides insight on a variation of the adaptability of a debilitated negative-strand RNA virus, namely the generation of defective minihelper viruses to overcome its restriction. This is in contrast to the common "defective interfering particles" that interfere with the replication of the virus from which they originated. To our knowledge, defective genomes that promote rather than inhibit replication have not been reported before in RNA viruses.

Keywords: codon-pair deoptimization; defective genomes; genetic stability; negative-strand RNA virus; respiratory syncytial virus.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Animals
  • Chlorocebus aethiops
  • Codon / genetics
  • Genome, Viral / genetics*
  • Open Reading Frames / genetics
  • Respiratory Syncytial Virus Vaccines / genetics*
  • Respiratory Syncytial Virus Vaccines / immunology
  • Respiratory Syncytial Virus, Human / genetics*
  • Respiratory Syncytial Virus, Human / immunology
  • Sequence Deletion
  • Vaccines, Attenuated / genetics
  • Vaccines, Attenuated / immunology
  • Vero Cells
  • Viral Fusion Proteins / genetics
  • Virus Replication / genetics*


  • Codon
  • Respiratory Syncytial Virus Vaccines
  • Vaccines, Attenuated
  • Viral Fusion Proteins