Sequence diversity of the reverse transcriptase of human immunodeficiency virus type 1 from untreated Brazilian individuals

Abstract

The presence of human immunodeficiency virus type 1 (HIV-1) bearing mutations resistant to nucleosidic inhibitors of the viral reverse transcriptase (RT) derived from HIV-seropositive asymptomatic and untreated volunteer blood donors was examined. The RT amplicons of 32 specimens were analyzed by using a reverse hybridization line probe assay technique that detects resistance against zidovudine (3'-azido-3'-deoxythymidine [AZT], didanosine (2',3'-dideoxyinosine [ddI], zalcitabine (2',3'-dideoxycytidine [ddC]), and lamivudine ((-)-beta-L-2',3'-dideoxy-3'-thiacytidine [3TC]) at amino acid positions 41, 69, 70, 74, 184, and 215 of the HIV RT. One sample (brp004, subtype B) showed an AZT resistance secondary mutation at position K70R. Fifteen specimens revealed one or more sites of nonreactivity to both wild-type- and mutant-specific probes (dual nonreactivity). Samples were also submitted to RT direct sequencing and phylogenetic analysis. Nine of 32 specimens belonged to non-B subtypes (C, D, F, and F/B or B/F mosaics). Three of these non-B isolates, named brp004, brp063, and brp069, revealed three other relevant AZT resistance mutations-a T215F mutation and two M41L mutations, respectively-hidden by the nonreactivity to line probe assay strips on the respective codon regions. The isolate brp004 also carried a D67N AZT resistance mutation revealed by direct sequencing. No nonnucleosidic RT inhibitor-resistant mutation was found. The analysis revealed a frequency of 2.26 x 10(-4) mutations per nucleotide for independent samples related to RT resistance. These findings emphasize the magnitude of naturally occurring reservoirs of drug-resistant virus among untreated HIV-1-positive individuals in Brazil.

FIG. 1

Phylogenetic analysis of the pol RT genes of 18 HIV isolates from Brazilian blood donors (A) compared to reference HIV-1 group M subtypes available in the Los Alamos database. Fragments containing a 571-nt region of the HIV RT gene were aligned for the generation of the phenogram. (B) The phenogram was performed without the alignment of the brp069 recombinant sequence. The RT sequence of SIVcpz was used as an outgroup, and bootstrap values for 100 replicates are listed at the major subtype branches. The brazilian isolates sequenced in this study are highlighted. Specimens of mixed subtype are designated as follows: ∗, type B/F mosaics (RT intragenic mosaic and/or p24^gag gp41^env mosaic); ∗∗, brp026 type B/F/B mosaic (p24^gag/pol RT/gp41^env gp41); ∗∗∗, brp069 type B/F/B intragenic RT chimera and type B/F p24^gag/gp41^env mosaic. The hash mark on SIVcpz indicates a truncation of actual distance.

FIG. 2

Sequence alignment of the RT gene (nt 106 to 676) of Brazilian specimens brp019 brp011 (subtype B); brp063 and brp093 (subtype B/F mosaics); brp069 (subtype B/F/B chimera); and brp034, brp035, and brp127 (subtype F). The prototypes HXB2 and OYI (subtype B) were also included, as well as the mosaic B/F sequence USAF, which was from an HIV isolate from an American subject undergoing antiretroviral treatment. The dots represent similarity of sequence; the arrows indicate the regions near the putative recombination sites at the brp069 RT gene. The highlighted and italicized bases represent the subtype F sequence signature found.