r-82150 and 2--3--dideoxyguanosine-5--triphosphate

Many bacterial expression systems have been developed to study the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). This enzyme exists in the virions as a heterodimer of a 66 kDa (p66) subunit and a 51 kDa (p51) subunit, originating through proteolytic maturation of the p66 subunit. Most expression systems rely on the processing of p66 by bacterial proteases, this results in a p51 subunit with a non-authentic carboxy-terminus. In contrast, the expression system described produces an RT with an authentic carboxy-terminus. This was achieved by the co-expression of the two subunits of HIV-1 RT, which were each cloned on a different, compatible plasmid in Escherichia coli, and by the use of protease inhibitors during cell lysis. This approach enabled us not only to obtain virion-like RT, as verified by mass spectrometry, but also to monitor the effect of mutations in one or both subunits on the activity of RT and on its sensitivity towards RT inhibitors. The co-expression system described represents a useful method to produce HIV-1 RT, both authentic and mutated, in quantities that allow large-scale studies on the functional organisation of the RT-subunits and the sensitivity of the enzyme to RT inhibitors.

Topics: Benzodiazepines; Deoxyguanine Nucleotides; Dideoxynucleotides; Escherichia coli; Gene Expression; HIV Reverse Transcriptase; HIV-1; Humans; Imidazoles; Mass Spectrometry; Plasmids; Recombinant Fusion Proteins; Reverse Transcriptase Inhibitors; Spiro Compounds; Thymidine; Virion

Determination of the three-dimensional structure of the human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) has indicated a totally different folding for the 51-kDa subunit (p51) than for the 66-kDa subunit (p66). The polymerase catalytic site is located on the p66 subunit. Moreover, the HIV-1-specific RT inhibitors, also designated as the non-nucleoside RT inhibitors (NNRTIs), select for amino acid mutations that afford resistance to these compounds and are clustered in the palm domain of the HIV-1 RT p66 subunit. This pocket is located in the vicinity of, but clearly distinct from, the polymerase active site. However, for the NNRTIs that belong to the class of the [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'',2''- oxathiole- 2'',2''-dioxide)] (TSAO) derivatives, the resistance mutation is located at position Glu138. On the p66 subunit, this amino acid is distant from the binding site of the HIV-1-specific RT inhibitors. When the TSAO-specific resistance mutation Glu138-->Lys was introduced solely in the p51 subunit of the RT p66/p51 heterodimer, the enzyme proved completely resistant to TSAO-m3T but retained full sensitivity to TIBO R82150 and ddGTP. On the other hand, when the mutation was introduced only in the p66 subunit the enzyme remained equally sensitive to the inhibitory effects of TSAO-m3T, TIBO R82150, and ddGTP. Our data provide compelling evidence for a structural and functional role of the p51 subunit in the sensitivity and/or resistance of the enzyme to the NNRTIs.

Topics: Antiviral Agents; Base Sequence; Benzodiazepines; Binding Sites; Deoxyguanine Nucleotides; Dideoxynucleotides; Drug Resistance; Escherichia coli; HIV Reverse Transcriptase; Imidazoles; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Protein Conformation; Recombinant Proteins; Reverse Transcriptase Inhibitors; RNA-Directed DNA Polymerase; Sequence Analysis, DNA; Spiro Compounds; Thymidine; Uridine

Recently, tetrahydroimidazo-[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione (TIBO) and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) compounds have been shown to be potent, selective, and specific inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in vitro. They interact with the reverse transcriptase of HIV-1 in a way different from that of previously studied reverse transcriptase (RT) inhibitors. We established an endogenous RT assay, starting from intact HIV-1 virions. This assay mimics the reverse transcription process in the HIV-infected cell more closely than RT assays with artificial templates. We investigated the inhibition of endogenous HIV-1 reverse transcription by the TIBO derivative (+)-(S)-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo [4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (R-82150) in comparison with the HEPT derivative 5-ethyl-1-ethoxymethyl-6-(phenylthio)uracil (E-EPU) and 2',3'-dideoxyguanosine 5'-triphosphate. The kinetics and characteristics of RT inhibition by TIBO in the endogenous RT assay were similar to those found previously for the exogenous RT assay (following addition of exogenous template/primer); thus, RT inhibition by TIBO was specific for HIV-1 and the extent of RT inhibition was dependent on which of the four substrates (dATP, dTTP, dGTP, and dCTP) was present in limited concentrations. Of the three enzymatic activities, RNA-dependent DNA polymerization was preferentially inhibited, and inhibition was not competitive with respect to the natural substrates. HIV-1 RT behaved as an allosteric enzyme, which means that positive cooperativity for binding of the substrate was observed. TIBO behaved as an allosteric inhibitor by causing a concentration-dependent decrease in this cooperativity.

Topics: Allosteric Regulation; Antiviral Agents; Base Sequence; Benzodiazepines; Cell Line; Deoxyguanine Nucleotides; Dideoxynucleotides; Electrophoresis, Agar Gel; HIV Reverse Transcriptase; HIV-1; Imidazoles; Kinetics; Molecular Sequence Data; Monocytes; Reverse Transcriptase Inhibitors; RNA-Directed DNA Polymerase; Thymine; Transcription, Genetic; Virus Replication