title: Quantitative SNP-detection method for estimating HIV-1 replicative fitness: application to protease inhibitor-resistant viruses.
abstract: We have improved the methods for the standard competitive growth assay of human immunodeficiency virus type 1 (HIV-1). The cloning step for the mixed viral population and subsequent genotype analysis for arbitrary numbers of clones were excluded from procedures. Instead, a single nucleotide polymorphism (SNP)-detection step was devised for the determination of viral populations. The quantitative SNP-detection method can rapidly estimate the proportion of wild-type and mutant populations with high reproducibility. Consequently, this method allows manipulation of many samples within a short period. Using this new competitive growth assay, replicative fitness of drug-resistant HIV-1 containing an M46I amino acid mutation in the protease was assessed in the presence or absence of indinavir. Without indinavir, replicative fitness of wild-type HIV-1 surpassed that of M46I-mutated HIV-1, and the fraction of mutated virus was reduced to about 10% at passage #9. In contrast, the fraction of M46I-mutated virus increased to >90% at passage #5 in the presence of 26.4 nM indinavir. Almost identical results were obtained for L90M-mutated HIV-1 with or without saquinavir. HIV-1 can survive under indinavir pressure by acquiring M46I mutation, as with acquisition of the L90M mutation under saquinavir pressure. However, these mutations damage viral replicative fitness under natural conditions without any drugs. Subtle differences between wild-type and mutant viruses are thus easily detected using the improved method.
authors.affiliation: Clinical Research Center, National Hospital Organization Nagoya Medical Center (Tokai Area Central Hospital for AIDS Treatment and Research), Sannomaru 4-1-1, Naka-ku, Nagoya, Aichi 460-0001, Japan.
authors.forename: Shiro
authors.initials: S
authors.lastname: Ibe
authors.forename: Saeko
authors.initials: S
authors.lastname: Fujisaki
authors.forename: Seiichiro
authors.initials: S
authors.lastname: Fujisaki
authors.forename: Takayuki
authors.initials: T
authors.lastname: Morishita
authors.forename: Tsuguhiro
authors.initials: T
authors.lastname: Kaneda
mesh_terms: D024882:Drug Resistance, Viral; D016333:HIV Protease / Q000235:genetics*; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D019469:Indinavir / Q000494:pharmacology*; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D014779:Virus Replication
chemical_list: D017320:HIV Protease Inhibitors; D019469:Indinavir; D016333:HIV Protease