Tryptophan scanning mutagenesis of aromatic residues within the polymerase domain of HIV-1 reverse transcriptase: critical role of Phe-130 for p51 function and second-site revertant restoring viral replication capacity.
The effects on virus viability and reverse transcriptase (RT) function of substituting Trp for Tyr or Phe residues within the polymerase domain of human immunodeficiency virus type 1 (HIV-1) RT have been analyzed with an infectious HIV-1 clone. Viruses containing mutations Y56W, F61W, F87W, F116W, Y127W, Y144W, F171W, Y181W, Y183W, Y188W, F227W, or Y232W in their RT-coding regions were viable and showed replication capacities similar or slightly reduced in comparison with the wild-type HIV-1. However, RTs bearing mutations F77W or Y146W had a dNTP-binding defect, rendering nonviable viruses. HIV-1 carrying RT mutations F124W or F130W replicated very poorly, but compensatory changes (K83R for F124W, and T58S for F130W) were selected upon passaging the virus in cell culture. The amino acid substitution F130W diminishes the stability of the 51-kDa subunit of the RT (p51) and impairs polyprotein processing in virus-infected cells, an effect that can be mitigated when T58S is found in p51.
Olivares
Isabel
I
Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
Gutiérrez-Rivas
Mónica
M
López-Galíndez
Cecilio
C
Menéndez-Arias
Luis
L
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015497:HIV-1 / Q000201:enzymology / Q000502:physiology*; D006801:Humans; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D013698:Templates, Genetic; D014364:Tryptophan / Q000737:chemistry; D014779:Virus Replication
D021122:Protein Subunits; D010649:Phenylalanine; D014364:Tryptophan; D054303:HIV Reverse Transcriptase; D012321:DNA-Directed RNA Polymerases