595 Identification of the Minimal Conserved Structure of the HIV-protease under the Presence and Absence of Drug Pressure F Ceccherini-Silberstein*1, A Bertoli2, F Forbici2, F Erba1, L Marcon3, S Aquaro1, C Balotta4, S Giannella2, R D'Arrigo2, MC Bellocchi2, C Gori2, MP Trotta2, A d'Arminio Monforte4, A Antinori2, CF Perno1,2 1Univ of Rome Tor Vergata, Italy; 2Inst Natl per le Malattie Infettive L Spallanzani, Rome, Italy; 3Univ of Padua, Italy; and 4Univ of Milan, Italy
Background: Extensive polymorphisms and drug-related variability occur in the HIV-1 protease (PR); however, little is known regarding its minimal conserved structure. The extent of PR conservation in vivo in the absence and presence of pharmacological pressure was then studied in a large cohort of patients (pts).
Methods: Sequences of the whole PR from plasma viral RNA samples of 413 drug-na´ve and 412 HAART (PR inhibitors [PI] included) treated pts were obtained and compared to a NL4.3 reference strain. The predicted tri-dimensional structure of the enzyme was then analyzed using Swiss PDB viewer v3.7 software.
Results: The PR amino acid (aa) sequence from drug-na´ve pts showed 2 large conserved regions (frequency of mutations < 1%) containing the catalytic active-site (E21-V32), the flap (P44-V56), and 8 small conserved domains (2-4aa each). In PI-treated pts, conserved regions shrunk and clustered to 9 small domains (including active-site D25-D29, and top of the flap G49-G52). The degree of conservation at some residues of the PR N- and C-terminal regions (both relevant for protein dimerization) was even higher in PI-treated pts. In addition to the emergence of primary and secondary mutations, 10 residues showed increased variability as "novel" mutations in treated pts (with p < 0.05 at codons A22, Q61, I66, Q92; and with p < 0.001 at codons K55, Q58, I62, I72, T74, I85). Among these novel mutations, K55R, Q58E, T74S and I85V were significantly (p < 0.05- < 0.001) associated, or mutually exclusive, to specific primary and secondary mutations (L10I, K20R, M36I, M46I, G48V, I54V, A71V, G73S, V82A, I84V, L90M, I93L). Overall, conserved aa in the whole protein sequences were 37/99 (< 0.5% variability); of them, 26 were highly-conserved (< 0.25% variability), with 11 residues totally unchanged. The highly-conserved aa were scattered or distributed in 5 small domains, that included the most relevant aa for the dimer stability (L5, T26, I50, R87, T91, T96-F99), the catalytic site D25-D29, the top of the flap G49-G52, the loop G78-P81, and the N- and C-terminal sites W6-P9, L97-F99.
Conclusions: In a frame of a wide polymorphism throughout its whole tertiary structure (that reflects the ability of the enzyme to adapt to drug pressure), selected areas of HIV-PR represent the minimal regions essential for the preservation of a functional and stable structure of the enzyme. Future new HIV-PR inhibitors should be designed to interfere with these invariant domains.