A positively charged side chain at position 154 on the beta8-alphaE loop of HIV-1 RT is required for stable ternary complex formation.

Lys154 is the only positively charged residue located in the VLPQGWK motif on the beta8-alphaE loop at the junction of the fingers and palm subdomains of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Some of the conserved residues in this motif are critical for RT function, while others have been shown to confer nucleoside drug resistance and fidelity to the enzyme. In order to understand the functional implication of this positively charged residue, we carried out site-directed mutagenesis at position 154 and biochemically characterized the mutant enzymes.

Substitution of conserved hydrophobic residues in motifs B and C of HIV-1 RT alters the geometry of its catalytic pocket.

Recent crystallographic data suggest that a number of hydrophobic residues seen clustered between the structurally conserved alphabetabetaalpha motif of the palm subdomain and at the junction of palm and fingers subdomains of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) provide an optimal geometry to the alphabeta sandwich of the palm subdomain, which harbors the catalytic site and the primer-binding grip region. This region has also been implicated in binding to the non-nucleoside RT inhibitors. We have evaluated the impact of conserved and nonconserved amino acid substitutions at four hydrophobic positions in this region of HIV-1 RT, in the context of their biochemical characteristics.

PNA targeting the PBS and A-loop sequences of HIV-1 genome destabilizes packaged tRNA3(Lys) in the virions and inhibits HIV-1 replication.

During assembly of the HIV-1 virions, cellular tRNA(Lys)(3) is packaged into the virion particles and is utilized as a primer for the initiation of reverse transcription. The 3'-terminal 18 nucleotides of the cellular tRNA(Lys)(3) are complementary to nucleotides 183-201 of the viral RNA genome, referred to as the primer binding sequence (PBS). Additional sequences (A-Loop) upstream of the PBS are essential for tRNA primer selection.

Lysine 152 of MuLV reverse transcriptase is required for the integrity of the active site.

Comparison of the three-dimensional structure of the active sites of MuLV and HIV-1 reverse transcriptases shows the presence of a lysine residue (K152) in the substrate-binding region in MuLV RT, while its equivalent position in HIV-1 RT is occupied by a glycine (G112). To investigate the role of K152 in the mechanism of the polymerase reaction catalyzed by MuLV RT, four mutant RTs, namely, K152A, K152R, K152E, and K152G, were generated and biochemically characterized. All muteins exhibited reduced polymerase activity on both RNA and DNA template-primers with K152E being the most defective.

Inhibition of HIV-1 replication by anti-trans-activation responsive polyamide nucleotide analog.

Efficient replication and gene expression of human immunodeficiency virus-1 (HIV-1) involves specific interaction of the viral protein Tat, with its trans-activation responsive element (TAR) which forms a highly stable stem-loop structure. We have earlier shown that a 15-mer polyamide nucleotide analog (PNA) targeted to the loop and bulge region of TAR blocks Tat-mediated transactivation of the HIV-1 LTR both in vitro and in cell culture (Mayhood et al., Biochemistry 39 (2000) 11532).

Insertion of a small peptide of six amino acids into the beta7-beta8 loop of the p51 subunit of HIV-1 reverse transcriptase perturbs the heterodimer and affects its activities.

Background: HIV-1 RT is a heterodimeric enzyme, comprising of the p66 and p51 subunits. Earlier, we have shown that the beta7-beta8 loop of p51 is a key structural element for RT dimerization (Pandey et al., Biochemistry 40: 9505, 2001).

Anti-TAR polyamide nucleotide analog conjugated with a membrane-permeating peptide inhibits human immunodeficiency virus type 1 production.

The emergence of drug-resistant variants has posed a significant setback against effective antiviral treatment for human immunodeficiency virus (HIV) infections. The choice of a nonmutable region of the viral genome such as the conserved transactivation response element (TAR element) in the 5' long terminal repeat (LTR) may potentially be an effective target for drug development. We have earlier demonstrated that a polyamide nucleotide analog (PNA) targeted to the TAR hairpin element, when transfected into cells, can effectively inhibit Tat-mediated transactivation of HIV type 1 (HIV-1) LTR (T.