The N137 and P140 amino acids in the p51 and the P95 amino acid in the p66 subunit of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase are instrumental to maintain catalytic activity and to design new classes of anti‐HIV‐1 drugs

Abstract

The N137 and P140 amino acids in the p51 and the P95 amino acid in the p66 subunit of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase are instrumental to maintain catalytic activity and to design new classes of anti-HIV-1 drugs Abstract Amino acids N137 and P140 in the p51 subunit of HIV-1 reverse transcriptase (RT) are part of the b7-b8-loop that contributes to the formation of the base of the non-nucleoside RT inhibitor (NNRTI)-binding pocket and makes up a substantial part of the dimerization interface. Amino acid P95 in p66 also markedly contributes to the dimerization binding energy. Nine RT mutants at amino acid 137 were constructed bearing the mutations Y, K, T, D, A, Q, S, H or E. The prolines at amino acid positions 95 and 140 were replaced by alanine in separate enzymes. We found that all mutant RT enzymes showed a dramatically decreased RNA-dependent DNA polymerase activity. None of the mutant RT enzymes showed marked resistance against any of the clinically used NNRTIs but they surprisingly lost significant sensitivity for NRTIs such as ddGTP. The denaturation analyses of the mutant RTs by urea are suggestive for a relevant role of N137 in the stability of the RT heterodimer and support the view that the b7-b8 loop in p51 is a hot spot for RT dimerization and instrumental for efficient polymerase catalytic activity. Consequently, N137 and P140 in p51 and P95 in p66 should be attractive targets in the design of new structural classes of RT inhibitors aimed at compromising the optimal interaction of the b7-b8 loop in p51 at the p66/p51 dimerization interface.

Structural and Preclinical Studies of Computationally Designed Non-Nucleoside Reverse Transcriptase Inhibitors for Treating HIV infection

“…Mutations with these two residues might destabilize the RT dimer or interfere with the correct placement of the primer in the active site, leading to a significant decrease in RT activity and therefore viral fitness. Indeed, clinical studies and in vitro mutational analysis have shown that both residues are immutable (Pelemans et al, 2000;Auwerx et al, 2005;Ceccherini-Silberstein et al, 2005), suggesting that compound II's interactions with these two residues will be able to compensate for potential lost interactions due to other NNBP mutations. Moreover, although compound II forms a hydrogen bond with the frequently mutated K103, the hydrogen bond donor is the backbone amide of K103, suggesting that the interaction is likely maintained in a number of K103 mutations.…”

HIV-1 Protease Dimer Interface Mutations that Compensate for Viral Reverse Transcriptase Instability in Infectious Virions

“…22 Several amino acid substitutions within the β7-β8 loop of p51 are known to impair heterodimer formation, while decreasing the DNA polymerase activity of the viral RT in enzymatic assays. [16][17][18] Molecular mechanisms leading to fitness recovery play an important role in antiretroviral therapy. Evolution of drug resistance is characterized by severe fitness losses, which can be partially overcome by compensatory mutations or other adaptive changes that restore viral replication capacity.…”

“…In p51, residues 52-55 and 135-140 in the fingers subdomain, 255-265 and 286-290 in the thumb subdomain, and 393-402 and 420-423 in the connection subdomain generate the largely hydrophobic surface that interacts with p66. 15 Several non-conservative mutations in p51 that affect residues of the β7-β8 loop (positions 134-139) [16][17][18][19] or the so-called "Trp motif" (residues 398, 401, 402, 406, 410 and 414) 20,21 are known to impair RT dimerization, while producing a loss of viral infectivity.…”

Basis for Early and Preferential Selection of the E138K Mutation in HIV-1 Reverse Transcriptase

“…Therefore, the 3= ultimate and penultimate positions of the AS primer were designed to overlap with the first two nucleotides of the E138 codon (see Table S1B in the supplemental material). Intentional mismatches were placed on two invariant adenine nucleotides of residue N137 (27,34,35) to increase the sensitivity. Of note, approximately half of subtype B and subtype C viruses should not have any mutations in the AS primer-binding site (27).…”