Journal
MICROBIOLOGY AND IMMUNOLOGY
Volume 50, Issue 10, Pages 765-772Publisher
WILEY
DOI: 10.1111/j.1348-0421.2006.tb03852.x
Keywords
quantitative SNP-detection; competitive growth assay; viral replicative fitness; protease inhibitor-resistant HIV-1
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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.
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