Winters and Merigan recently reported interesting results showing that several amino acid substitutions encoded by codon 69 of the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) gene affect susceptibility to nucleoside-nucleotide analog RT inhibitors (NRTIs) (5). While it is classically considered that the T69D/N mutation confers resistance to zalcitabine (dideoxycytidine [ddC]) (1), we have demonstrated, and Winters and Merigan have confirmed, that this mutation can be identified in ddC-naive individuals (4). On the basis of this observation, we have further analyzed the amino acid substitutions at position 69 in the RT sequences obtained routinely in our laboratory; amino acid insertions or deletions at this position have not been considered in this study.
After exclusion of the patients tested after treatment interruption, 789 RT sequences were available for analysis: 34 from antiretroviral-naive patients and 755 from antiretroviral-experienced patients. Among the antiretroviral-experienced patients, 50 (6.6%) had received one antiretroviral regimen, 444 (58.8%) had received two or three antiretroviral regimens, and 261 (34.6%) had received four or more antiretroviral regimens; 3 (0.4%) had received one NRTI, 58 (7.7%) had received two NRTIs, 89 (11.8%) had received three NRTIs, 221 (29.3%) had received four NRTIs, 235 (31.1%) had received five NRTIs, 145 (19.2%) had received six NRTIs, and 4 (0.5%) had received seven NRTIs.
Amino acid substitutions at position 69 were not found in sequences from the NRTI-naive patients but were found in those from 116 (15.4%) NRTI-experienced patients: 71 patient sequences (61.2%) had T69D mutations, 28 (24.1%) had T69N mutations, 7 (6.0%) had T69S mutations, 4 (3.4%) had T69A mutations, 3 (2.6%) had T69E mutations, and 1 each (0.9%) had T69G, T69I, and T69L mutations.
The prevalence of amino acid substitutions at position 69 increased significantly with the number of drug regimens that a patient received (P = 0.0002), since a prevalence of 8.0% was observed in patients who had received one treatment regimen and a prevalence of 21.8% was observed in patients who had received four or more treatment regimens. A significant relationship (P = 0.01) between the prevalence of amino acid substitutions at position 69 and the number of NRTIs that a patient received was also observed.
A multivariate logistic-regression analysis showed that the number of antiretroviral drug regimens that a patient received was the sole factor independently related to amino acid substitutions at position 69. However, when only the D and N substitutions were considered, a significant relationship (P = 0.03) between the T69D/N mutation and experience with didanosine (ddI) was observed (data not shown). There was no significant relationship between D/N (P = 0.26) or other amino acid substitutions (P = 0.13) at position 69 and experience with ddC.
By univariate analysis, amino acid substitutions at position 69 were significantly associated with mutations at positions 44, 67, 70, 118, 210, 215, and 219, whereas a borderline significance was observed for the association with the L74V mutation (Table 1). By multivariate analysis, however, only E44D/A (P = 0.02), D67N (P < 0.0001), and V118I (P = 0.0001) remained significantly associated with amino acid substitutions at position 69. By focusing on the D and N substitutions, a significant association was observed with the T69D/N mutation and the Q151M mutation (P = 0.004).
TABLE 1.
Association of the amino acid substitutions at position 69 with other NRTI resistance mutations in 789 sequences
| Amino acid and position | Substituted amino acida | Prevalence of substitutionsd(%) | Pb |
|---|---|---|---|
| M41 | L (n = 412) | 16.2 | |
| None (WTc) | 13.0 | 0.19 | |
| E44 | D/A (n = 128) | 28.9 | |
| None (WT) | 11.9 | <0.0001 | |
| D67 | N (n = 331) | 27.5 | |
| None (WT) | 5.7 | <0.0001 | |
| K70 | R (n = 173) | 28.3 | |
| None (WT) | 10.9 | <0.0001 | |
| L74 | V (n = 99) | 21.2 | |
| None (WT) | 13.7 | 0.05 | |
| V75 | M/S/A/T (n = 54) | 16.6 | |
| None (WT) | 14.5 | 0.67 | |
| V118 | I (n = 194) | 29.4 | |
| None (WT) | 9.9 | <0.0001 | |
| Q151 | M (n = 31) | 19.3 | |
| None (WT) | 14.5 | 0.45 | |
| M184 | V/I (n = 404) | 15.0 | |
| None (WT) | 14.3 | 0.74 | |
| L210 | W (n = 278) | 18.7 | |
| None (WT) | 12.5 | 0.02 | |
| T215 | Y/F (n = 472) | 18.0 | |
| None (WT) | 9.8 | 0.001 | |
| K219 | Q/E (n = 166) | 28.9 | |
| None (WT) | 10.9 | <0.0001 |
n, number of sequences.
Chi-square test; a P value of <0.05 is considered significant.
WT, wild type.
Codon 69.
These results indicate that amino acid substitutions (mainly T→D and T→N) are common in multi-NRTI-experienced patients, that the prevalence of these mutations increases with the number of NRTI combination regimens received, and that previous ddI rather than ddC experience could be a factor of selection of the T69D/N mutations in multi-NRTI-treated pa-tients. These conditions of selection are also supported by the association observed between amino acid substitutions at position 69 and the E44A/D, V118I, and Q151M mutations, which are also known to be associated with multiple NRTI experience, including experience with ddI (2, 3). Amino acid substitutions were also closely associated with the D67N mutation, which is located in the same β3-β4 hairpin loop (positions 67 to 70) region of the RT; this association suggests that interactions exist between these mutations.
Our results, in agreement with those reported by Winters and Merigan (5), indicate that amino acid substitutions at position 69 may be selected for by ddC-free antiretroviral regimens. These mutations could therefore be involved in resistance to NRTIs other than ddC. This is supported by the findings of Winters and Merigan that show that different substitutions at position 69 confer reduced susceptibility to zidovudine, ddI, and stavudine.
The T69D/N mutation is currently taken into consideration for interpreting resistance to ddI (low-level resistance) and stavudine (potential low-level resistance) by using the Stanford HIV RT and protease sequence database algorithm (http://hivdb.stanford.edu). However, more effort is needed to accurately interpret HIV-1 genotypes that encode amino acid sub-stitutions at position 69. In particular, the effect of the different amino acid substitutions at this position and the interactions of these substitutions with the other NRTI resistance-associated mutations remain poorly documented.
Ed. Note: The authors of the published article declined to respond.
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