ABSTRACT
While many point mutations in the HIV-1 reverse transcriptase (RT) confer resistance to antiretroviral drugs, inserts or deletions in this gene have not been previously characterized. In this report, 14 RT inhibitor-treated patients were found to have HIV-1 strains possessing a 6-basepair insert between codons 69 and 70 of the RT gene. Known drug resistance mutations were also observed in these strains, with T215Y appearing in all strains. Genotypic analysis indicated that the inserts had substantial nucleotide variability that resulted in relatively restricted sets of amino acid sequences. Linkage of patients' treatment histories with longitudinal sequencing data showed that insert strains appeared during drug regimens containing ddI or ddC, with prior or concurrent AZT treatment. Drug susceptibility tests of recombinant patient isolates showed reduced susceptibility to nearly all nucleoside RT inhibitors. Site- directed mutagenesis studies confirmed the role of the inserts alone in conferring reduced susceptibility to most RT inhibitors. The addition of AZT-associated drug resistance mutations further increased the range and magnitude of resistance. These results establish that inserts, like point mutations, are selected in vivo during antiretroviral therapy and provide resistance to multiple nucleoside analogs.
Subject(s)
Drug Resistance, Multiple/genetics , HIV Reverse Transcriptase/genetics , HIV-1/genetics , Nucleosides/antagonists & inhibitors , Amino Acid Sequence , Anti-HIV Agents/therapeutic use , Base Sequence , Cells, Cultured , Didanosine/therapeutic use , HIV Infections/drug therapy , Humans , Mutagenesis, Site-Directed , Mutation , Reverse Transcriptase Inhibitors/therapeutic use , Zalcitabine/therapeutic use , Zidovudine/therapeutic useABSTRACT
Human immunodeficiency virus type 1 (HIV-1) pol mutations are responsible for HIV-1 resistance to current antiretroviral drugs. HIV-1 RNA extraction with QIAamp HCV kit spin columns (Qiagen, Chatsworth, Calif.) followed by reverse transcription-PCR successfully recovered a 1,008-bp pol fragment from the plasma of 31 of 34 HIV-1-infected patients that was suitable for sequencing and recombinant-virus studies. The minimum HIV-1 RNA concentration required for gene recovery was 30 to 40 copies/ml, which was similar to the minimal HIV-1 RNA concentration required when phenol-chloroform or silica beads are used for RNA extraction.
