Changes in codon-pair bias of human immunodeficiency virus type 1 have profound effects on virus replication in cell culture

• Autores: Glòria Martrus Zapater
• Directores de la Tesis: Josefa Badia Palacín (dir. tes.), Miguel Ángel Martínez de la Sierra (dir. tes.)
• Lectura: En la Universitat de Barcelona ( España ) en 2013
• Idioma: inglés
• Tribunal Calificador de la Tesis: Esteban Domingo Solans (presid.), Rosa María Pintó Solé (secret.), M.eloisa Yuste Herranz (voc.)
• Materias:
  • Ciencias de la vida
    • Virología
  • Ciencias médicas
    • Medicina interna
      • Enfermedades infecciosas
• Enlaces
  • Tesis en acceso abierto en:  TDX  Dipòsit Digital de la UB 
• Resumen

  • Human immunodeficiency virus type 1 (HIV-1) has a biased nucleotide composition different from human genes. This raises the question of how evolution has chosen the nucleotide sequence HIV-1 observed today, or to what extent the actual encoding contributes to virus replication capacity, evolvability and pathogenesis. Prior work has documented the effectiveness of making changes to the codon-pair bias of viral genomes in order to generate attenuated poliovirus and influenza virus. In this thesis, we applied the previously described synthetic attenuated virus engineering (SAVE) approach to HIV-1. Using synonymous codon pairs, we rationally recoded and codon pair–reoptimized and deoptimized different moieties of the HIV-1 gag and pol genes. RNA structures and codon usage of new recoded fragments were not affected by recoding. Deoptimized viruses had significantly lower viral replication capacity in MT-4 cells and peripheral blood mononuclear cells (PBMCs). Various degrees of ex vivo attenuation were obtained depending upon the specific deoptimized region and the number of deoptimized codons. After restricting viral replication to a single cycle by using a single-cycle HIV-1 vector, a significant reduction in protein production was observed in the vector carrying an attenuated virus variant. This reduction in protein synthesis was not accompanied by a reduction in the targeted transcript copy number, which strongly suggests that translation, and not transcription, is implicated in the generation of the attenuated phenotype by SAVE technology. A protease reoptimized virus carrying 38 synonymous mutations was not attenuated and displayed a replication capacity similar to that of the wild type virus in MT-4 cells and PBMCs. Although attenuation is based on several tens of nucleotide changes, after serial passages in MT-4 cells, both gag and protease deoptimized HIV-1 reverted to wild-type virulence in MT-4 cells while some maintain a certain attenuation degree in PBMCs. Quasispecies analysis of viral passaged sequences showed that attenuated viruses accumulated either synonymous mutations (reversions to wild-type sequences or novel mutations) or non-synonymous mutations. Recoded viruses explored different space sequences. Remarkably, no important reversion was observed in the reoptimized virus. Thus, these data demonstrate that SAVE is a useful strategy to gradually affect the replicative properties of HIV-1 by a mechanism that involves translation. HIV-1 with different degrees of attenuation can be a useful tool for the development of a safe and effective vaccine as well as the development of safer gene-therapy lentiviral vectors.