Genetic diversity of protease and reverse transcriptase sequences in non-subtype-B human immunodeficiency virus type 1 strains: evidence of many minor drug resistance mutations in treatment-naive patients

Abstract

Most human immunodeficiency virus (HIV) drug susceptibility studies have involved subtype B strains. Little information on the impact of viral diversity on natural susceptibility to antiretroviral drugs has been reported. However, the prevalence of non-subtype-B (non-B) HIV type 1 (HIV-1) strains continues to increase in industrialized countries, and antiretroviral treatments have recently become available in certain developing countries where non-B subtypes predominate. We sequenced the protease and reverse transcriptase (RT) genes of 142 HIV-1 isolates from antiretroviral-naive patients: 4 belonged to group O and 138 belonged to group M (9 subtype A, 13 subtype B, 2 subtype C, 5 subtype D, 2 subtype F1, 9 subtype F2, 4 subtype G, 5 subtype J, 2 subtype K, 3 subtype CRF01-AE, 67 subtype CRF02-AG, and 17 unclassified isolates). No major mutations associated with resistance to nucleoside reverse transcriptase inhibitors (NRTIs) or protease inhibitors were detected. Major mutations linked to resistance to non-NRTI agents were detected in all group O isolates (A98G and Y181C) and in one subtype J virus (V108I). In contrast, many accessory mutations were found, especially in the protease gene. Only 5.6% of the 142 strains, all belonging to subtype B or D, had no mutations in the protease gene. Sixty percent had one mutation, 22.5% had two mutations, 9.8% had three mutations, and 2.1% (all group O strains) had four mutations. In order of decreasing frequency, the following mutations were identified in the protease gene: M36I (86.6%), L10I/V (26%), L63P (12.6%), K20M/R (11.2%), V77I (5.6%), A71V (2.8%), L33F (0.7%), and M46I (0.7%). R211K, an accessory mutation associated with NRTI resistance, was also observed in 43.6% of the samples. Phenotypic and clinical studies are now required to determine whether multidrug-resistant viruses emerge more rapidly during antiretroviral therapy when minor resistance-conferring mutations are present before treatment initiation.

FIG. 1

Unrooted phylogenetic trees of protease and RT nucleotide sequences (1,600 bp) from 138 group M isolates (in gray) and from reference strains (in black) representing the different subtypes. The trees were generated as described in Materials and Methods. The reference sequences (8) used in the trees were as follows: A.U455, A.Q2317, A.92UG037.1, B.OYI, B.HXB2, B.JRFL, B.RF, D.NDK, D.ELI, D.84ZR085, D.94UG114.1, C.ETH2220, C.92BR025.8, F1.93BR020.1, F1.FIN9363, G.SE6165, G.92NG083, G(A).92NG003, G.HH8793.1.1, H.VI991, H.VI997, H.90CF056.1, J.SE9173.3, J.SE9280.9, CRF01-AE.CM240, CRF01-AE.93TH253.3, CRF01-AE.90CF402.1, CRF02-AG.IBNG, and CRF02-AG.DJ263. The reference sequences for the K and F2 subtypes (41, 42) were used either as references or as samples (K.96CAM.MP535, K.97ZR.EQTB11, F2.95CM.MP255, F2.95CM.MP257). For sequences that did not cluster with high bootstrap values with any of the known subtypes or CRFs, the results of complementary analyses indicating their recombinant nature are shown. ^a, the isolates designated A/G and G/A/G are intersubtype A/G recombinant viruses in which the breakpoints between A and G are different from those for the prototype CRF02-AG strain. The numbers at the branch points indicate bootstrap values as percentages.

FIG. 2

Amino acid alignment of protease consensus sequences for each subtype of group M and for group O. The sequences were aligned against a subtype B consensus sequence from the database; dots indicate homology. The amino acid positions associated with drug resistance are depicted in boldface italics; the major mutations (D30N, G48V, I50V, V82A/F/T, I84V, L90M) are marked at the top of the consensus sequence (▾), as are the minor mutations (L10I/V, K20M/R, L24I, V32I, L33F, M36I, M46I/L, I47V, I54L/V, L63P, A71T/V, G73S, V77I, N88D) (▿). The functional domains of the protease (∗, active site [amino acids 22 to 34]; ∗∗, flap region [amino acids 47 to 56]; ∗∗∗, substrate binding site [amino acids 78 to 88]) are shown under the consensus sequence.