RESUMEN
Substitution of particular residues postulated to have a role in active site architecture can alter the overall fidelity of DNA polymerization by HIV-1. The effects of this kind of substitution were determined in a lacZ-based assay using HIV-1 reverse transcriptase with specifically mutated residues. We found that the reported higher fidelity of nucleotide incorporation by the Met184-->Val and Glu89-->Gly mutant reverse transcriptases (RTs) was not reflected in a substantial increase in the overall fidelity for these RT mutants. For the 3TC-resistant Met184-->Val RT mutant an almost wild-type level of overall mutation frequency was observed, while the foscarnet-resistant RTs harbouring the Glu89-->Gly mutation showed only a twofold decrease in mutation frequency. The Tyr183-->Phe mutant RT displayed a slightly lower fidelity than wild-type RT. Conversely, the ddI-resistant RT mutant containing the Leu74-->Val mutation showed a 3.5-fold higher fidelity compared to the wild-type enzyme. Finally, the Tyr115-->Ala substitution rendered the enzyme substantially more error-prone for DNA polymerization. These results correlate with three-dimensional structural studies of the polymerase active site and confirm the postulated impact of the Leu74, Tyr183 and Tyr115 RT residues on the overall fidelity of DNA polymerization by HIV-1 RT.
Asunto(s)
Aminoácidos/química , Transcriptasa Inversa del VIH/metabolismo , Sustitución de Aminoácidos , Aminoácidos/genética , Secuencia de Bases , Biopolímeros , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Homología de Secuencia de Ácido NucleicoRESUMEN
Since the Human Immunodeficiency Virus Type 1 (HIV-1) was identified as the etiologic agent of the Acquired Immune Deficiency Syndrome (AIDS), the HIV-1 reverse transcriptase (RT) has been the subject of intensive study. The reverse transcription entails the transition of the single-stranded viral RNA into double-stranded proviral DNA, which is then integrated into the host chromosome. Therefore, the HIV-1 reverse transcriptase plays a pivotal role in the life cycle of the virus and is consequently an interesting target for anti-HIV drug therapy. In the first section, we describe the complex process of reverse transcription and the different activities involved in this process. We then highlight the structure-function relationship of the HIV-1 reverse transcriptase, which is of great importance for a better understanding of resistance development, a major problem in anti-AIDS therapies. Finally, we summarize the mechanisms of HIV resistance toward various RT inhibitors and the implications thereof for the current anti-HIV drug therapies.
Asunto(s)
Transcriptasa Inversa del VIH/metabolismo , Transcripción Genética , Fármacos Anti-VIH/farmacología , Farmacorresistencia Microbiana/genética , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Conformación Proteica , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-ActividadRESUMEN
The highly conserved Tyr-318 is part of the non-nucleoside reverse transcriptase inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type I reverse transcriptase (RT) and makes contact within 4 A with the NNRTIs in all reported RT/NNRTI complexes. Using site-directed mutagenesis, six mutant RTs were constructed bearing the mutations Y318H, Y318K, Y318L, Y318C, Y318W, and Y318F. We found that only the Y318W and Y318F mutant RTs retained substantial RT activity, whereas the catalytic activities of the Y318K, Y318C, Y318H, and Y318L RT mutants were less than 5% of the wild-type activity. The Y318F mutant RT retained substantial sensitivity to the majority of NNRTIs tested, whereas the Y318W mutant RT showed varying degrees of resistance to NNRTIs. Subunit-specific site-directed mutagenesis revealed that there was no difference in the catalytic activity or resistance/sensitivity spectrum toward NNRTIs regardless of whether the Tyr-318 mutation was introduced in both subunits or only in the p66 subunit of RT. Recombinant viruses harboring the Y318F or Y318W mutation in the RT showed a similar resistance/sensitivity pattern to NNRTIs as their corresponding 318 mutant recombinant RTs. Our findings stress a functional or structural role for Tyr-318 in wild-type RT and argue for the design of novel NNRTIs that interact more closely with this amino acid in the NNRTI-specific pocket of human immunodeficiency virus type I RT.
Asunto(s)
Delavirdina/metabolismo , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , VIH-1/efectos de los fármacos , Conformación Proteica , Inhibidores de la Transcriptasa Inversa/metabolismo , Tirosina , Sustitución de Aminoácidos , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Delavirdina/química , Transcriptasa Inversa del VIH/genética , VIH-1/enzimología , VIH-1/genética , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Inhibidores de la Transcriptasa Inversa/químicaRESUMEN
We compared the development of resistance toward BI-RG-587 (nevirapine) and alpha-APA R89439 (loviride) starting from the wild-type HIV-1 strain IIIB and the 3TC-resistant HIV-1 strain containing the M184V mutation. The reverse transcriptase of the M184V mutant has been reported to have a higher fidelity. Our experiments showed that there was no significant delay in virus breakthrough of the M184V mutant as compared with the wild-type virus. We therefore conclude that the reported higher fidelity of the M184V mutant does not lead to a delay in the development of resistance to the nonnucleoside reverse transcriptase inhibitors nevirapine and loviride.
Asunto(s)
Fármacos Anti-VIH/farmacología , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , Lamivudine/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Acetamidas/farmacología , Acetofenonas/farmacología , Células Cultivadas , Efecto Citopatogénico Viral , Farmacorresistencia Microbiana , Genes Virales , VIH-1/enzimología , VIH-1/genética , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Nevirapina/farmacología , Análisis de Secuencia de ADN , Replicación Viral/efectos de los fármacosRESUMEN
We report the development of a new group of nonnucleoside reverse transcriptase inhibitors (NNRTIs). One of the most active congeners of this series of 1,1,3-trioxo-2H,4H-thieno[3,4-e] [1,2,4]thiadiazine (TTD) derivatives, i.e., 2-(3-fluorobenzyl)-4-cyanomethylen-l,1,3-trioxo-2H,4H- thieno [3,4-e] [1,2,4] thiadiazine) (QM96639) was found to inhibit human immunodeficiency virus (HIV) type 1 [HIV-1 (IIIB)] replication in MT-4 cells at a concentration of 0.09 microM. This compound was toxic for the host cells only at a 1,400-fold higher concentration. The TTD derivatives proved effective against a variety of HIV-1 strains, including those that are resistant to 3'-azido-3'-deoxythymidine (AZT), but not against HIV-2 (ROD) or simian immunodeficiency virus (SIV/ MAC251). HIV-1 strains containing the L100I, K103N, V106A, E138K, Y181C, or Y188H mutations in their reverse transcriptase (RT) displayed reduced sensitivity to the compounds. Their cross-resistance patterns correlated with that of nevirapine. 2-Benzyl-4-cyanomethylen-1,1,3-trioxo-2H,4H-thieno[3,4-e] [1,2,4]thiadiazine (QM96521) enhanced the anti-HIV-1 activity of AZT and didanosine in a subsynergistic manner. HIV-1-resistant virus containing the V179D mutation in the RT was selected after approximately six passages of HIV-1 (IIIB) in CEM cells in the presence of different concentrations of QM96521. From structure-activity relationship analysis of a wide variety of TTD derivatives, a number of restrictions appeared as to the chemical modifications that were compatible with anti-HIV activity. Modelling studies suggest that in contrast to most other NNRTIs, but akin to nevirapine, QM96521 does not act as a hydrogen bond donor in the RT-drug complex.
Asunto(s)
Fármacos Anti-VIH/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Nevirapina/farmacología , Fármacos Anti-VIH/química , Células Cultivadas/efectos de los fármacos , Didanosina/farmacología , Farmacorresistencia Microbiana , Transcriptasa Inversa del VIH/efectos de los fármacos , Humanos , Modelos Moleculares , Nevirapina/química , Relación Estructura-Actividad , Zidovudina/farmacologíaRESUMEN
Various analogues of adenosine have been described as inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, and some of these AdoHcy hydrolase inhibitors (e.g., 3-deazaadenosine, 3-deazaaristeromycin, and 3-deazaneplanocin A) have also been reported to inhibit the replication of human immunodeficiency virus type 1 (HIV-1). When evaluated against HIV-1 replication in MT-4 cells, macrophages, or phytohemagglutinin-stimulated peripheral blood lymphocytes infected acutely or chronically with HIV-1IIIB or HIVBaL strains, a wide range of adenosine analogues did not inhibit HIV-1IIIB replication for 50% at subtoxic concentrations. However, they inhibited HIV-1 replication in HeLa CD4+ LTR-LacZ cells at concentrations well below cytotoxicity threshold. A close correlation was found among the inhibitory effect of the compounds on AdoHcy hydrolase activity, their inhibition of HIV-1 replication in Hela CD4+ LTR-LacZ cells, and their inhibition of the HIV-1 Tat-dependent and -independent transactivation of the long terminal repeat, whereas no inhibitory effect was seen on HIV-1 reverse transcription or a Tat-independent cytomegalovirus promoter. Our results suggest that AdoHcy hydrolase and the associated S-adenosylmethionine-dependent methylation mechanism play a role in the process of long terminal repeat transactivation and, hence, HIV replication.
Asunto(s)
Fármacos Anti-VIH/farmacología , Inhibidores Enzimáticos/farmacología , Duplicado del Terminal Largo de VIH , VIH-1/efectos de los fármacos , VIH-1/fisiología , Hidrolasas/antagonistas & inhibidores , Activación Transcripcional/efectos de los fármacos , Proteínas Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Adenosilhomocisteinasa , Línea Celular , Productos del Gen tat/fisiología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/enzimología , Células HeLa/virología , Humanos , Hidrolasas/fisiología , Inhibidores de la Transcriptasa Inversa/farmacología , Proteínas Virales/fisiología , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
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.
Asunto(s)
Escherichia coli/metabolismo , Expresión Génica , Transcriptasa Inversa del VIH/genética , VIH-1/enzimología , Benzodiazepinas/farmacología , Nucleótidos de Desoxiguanina/farmacología , Didesoxinucleótidos , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/química , Humanos , Imidazoles/farmacología , Espectrometría de Masas , Plásmidos , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Inhibidores de la Transcriptasa Inversa/farmacología , Compuestos de Espiro/farmacología , Timidina/análogos & derivados , Timidina/farmacología , ViriónRESUMEN
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.