Background:  Polymorphisms and drug-related variability occur in HIV-1 reverse transcriptase (RT), however little is known regarding its minimal conserved structure. Therefore, the extent of RT conservation in vivo in absence and presence of drug pressure has been studied in a large cohort of patients.

Methods:  Plasma-derived complete sequences of the first 320 amino acids of RT from a well-defined cohort of 1259 patients (802 heavily drug-treated and 457 drug-naïve) were obtained and analysed. Positions with variability <1% were considered invariant.

Results:  In naïve patients, RT protein sequence showed no variation in 230/320 amino acids (72% overall conservation). Some invariant residues were scattered throughout the sequence either individually or in pairs, whereas others clustered into defined regions comprising 4 to 25 consecutive invariant residues. The 8 longest invariant regions were: I (T7-P19), II (W71-K82), III (T107-S117, containing the catalytic D110), IV (F124-S134), V (N147-P157), VI (Y181-E194, containing the catalytic D185-D186), VII (K220-I244), and VIII (W252-Y271). The frequency of mutations associated with drug-resistance was always <1% in drug-naïve patients. In treated patients, despite the appearance of mutations primarily associated with resistance, amino acid invariance was still maintained in 204/320 amino acids (64% of conservation vs 72% in naïve patients). The long conserved areas I, III, IV, V, and VIII were preserved in drug-treated pts, with minor exceptions of K11, V108, F116, Q151 (variability in <3% pts). Three other large conserved areas (II, VI, VII) shrank to isolated residues and smaller domains (W71-K73, R78-K82, D185-L187, S191-L193, W229-D237, W239-I244). Nucleotide sequence analysis in RT and protease showed a rate of synonymous/non-synonymous mutations of 7.7 and 4, respectively, thus suggesting a greater conservation of RT even under drug pressure. In addition, 12 new RT mutations were identified as positively (K20R, T39A, K43E, K43Q, K122E, E203K, D218E, Q278H, R284K, T286A) or negatively (R83K, V276I) associated with major RT-mutations and with treatment of RT inhibitors (p range <0.05 to <0.001).

Conclusions:  Even in drug-treated patients, HIV-1 RT requires the full preservation of at least 2/3 of its amino acids (some with still unknown function), and of large areas of its tertiary structure, to maintain a stable and functional enzyme. Future RT inhibitors may be designed to preferentially target these invariant domains.

Keywords: HIV-1 Reverse Transcriptase; conservation; structure