RESUMEN
HIV evades eradication because transcriptionally dormant proviral genomes persist in long-lived reservoirs of resting CD4(+) T cells and myeloid cells, which are the source of viral rebound after cessation of antiretroviral therapy. Dormant HIV genomes readily produce infectious virus upon cellular activation because host transcription factors activated specifically by cell stress and heat shock mediate full-length HIV transcription. The molecular chaperone heat shock protein 90 (Hsp90) is overexpressed during heat shock and activates inducible cellular transcription factors. Here we show that heat shock accelerates HIV transcription through induction of Hsp90 activity, which activates essential HIV-specific cellular transcription factors (NF-κB, NFAT, and STAT5), and that inhibition of Hsp90 greatly reduces gene expression mediated by these factors. More importantly, we show that Hsp90 controls virus transcription in vivo by specific Hsp90 inhibitors in clinical development, tanespimycin (17-(allylamino)-17-demethoxygeldanamycin) and AUY922, which durably prevented viral rebound in HIV-infected humanized NOD scid IL-2Rγ(-/-) bone marrow-liver-thymus mice up to 11 weeks after treatment cessation. Despite the absence of rebound viremia, we were able to recover infectious HIV from PBMC with heat shock. Replication-competent virus was detected in spleen cells from these nonviremic Hsp90 inhibitor-treated mice, indicating the presence of a tissue reservoir of persistent infection. Our novel findings provide in vivo evidence that inhibition of Hsp90 activity prevents HIV gene expression in replication-competent cellular reservoirs that would typically cause rebound in plasma viremia after antiretroviral therapy cessation. Alternating or supplementing Hsp90 inhibitors with current antiretroviral therapy regimens could conceivably suppress rebound viremia from persistent HIV reservoirs.
Asunto(s)
Infecciones por VIH/prevención & control , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Latencia del Virus/efectos de los fármacos , Animales , Antivirales/farmacología , Benzoquinonas/farmacología , Western Blotting , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Femenino , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas Macrocíclicas/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Like normal cellular nucleosides, the nucleoside reverse transcriptase (RT) inhibitor (NRTI) 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) has a 3'-hydroxyl moiety, and yet EFdA is a highly potent inhibitor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication with activity against a broad range of clinically important drug-resistant HIV isolates. We evaluated the anti-HIV activity of EFdA in primary human cells and in HIV-infected humanized mice. EFdA exhibited excellent potency against HIVJR-CSF in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs), with a 50% inhibitory concentration of 0.25 nM and a selectivity index of 184,000; similar antiviral potency was found against 12 different HIV clinical isolates from multiple clades (A, B, C, D, and CRF01_AE). EFdA was readily absorbed after oral dosing (5 mg/kg of body weight) in both mice and the rhesus macaque, with micromolar levels of the maximum concentration of drug in serum (Cmax) attained at 30 min and 90 min, respectively. Trough levels were at or above 90% inhibitory concentration (IC90) levels in the macaque at 24 h, suggesting once-daily dosing. EFdA showed reasonable penetration of the blood-brain barrier in the rhesus macaque, with cerebrospinal fluid levels at approximately 25% of plasma levels 8 h after single oral dosing. Rhesus PBMCs isolated 24 h following a single oral dose of 5 mg/kg EFdA were refractory to SIV infection due to sufficiently high intracellular EFdA-triphosphate levels. The intracellular half-life of EFdA-triphosphate in PBMCs was determined to be >72 h following a single exposure to EFdA. Daily oral administration of EFdA at low dosage levels (1 to 10 mg/kg/day) was highly effective in protecting humanized mice from HIV infection, and 10 mg/kg/day oral EFdA completely suppressed HIV RNA to undetectable levels within 2 weeks of treatment.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Compuestos de Diazonio/uso terapéutico , Farnesol/análogos & derivados , Infecciones por VIH/tratamiento farmacológico , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Animales , Fármacos Anti-VIH/administración & dosificación , Barrera Hematoencefálica , Compuestos de Diazonio/administración & dosificación , Compuestos de Diazonio/farmacocinética , Farnesol/administración & dosificación , Farnesol/farmacocinética , Farnesol/uso terapéutico , Citometría de Flujo , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Semivida , Humanos , Técnicas In Vitro , Macaca mulatta , Ratones , Ratones SCID , Monocitos/efectos de los fármacos , Monocitos/virología , ARN Viral/biosíntesis , ARN Viral/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Inhibidores de la Transcriptasa Inversa/farmacocinética , Virus de la Inmunodeficiencia de los Simios , Viremia/tratamiento farmacológico , Viremia/virologíaRESUMEN
Human cytomegalovirus (HCMV) is the leading viral cause of birth defects and life-threatening lung-associated diseases in premature infants and immunocompromised children. Although the fetal lung is a major target organ of the virus, HCMV lung pathogenesis has remained unexplored, possibly as a result of extreme host range restriction. To overcome this hurdle, we generated a SCID-hu lung mouse model that closely recapitulates the discrete stages of human lung development in utero. Human fetal lung tissue was implanted into severe combined immunodeficient (CB17-scid) mice and inoculated by direct injection with the VR1814 clinical isolate of HCMV. Virus replication in the fetal lung was assessed by the quantification of infectious virus titers and HCMV genome copies and the detection of HCMV proteins by immunohistochemistry and Western blotting. We show that HCMV efficiently replicated in the lung implants during a 2-week period, forming large viral lesions. The virus productively infected alveolar epithelial and mesenchymal cells, imitating congenital infection of the fetal lung. HCMV replication triggered apoptosis near and within the viral lesions and impaired the production of surfactant proteins in the alveolar epithelium. Our findings highlight that congenital and neonatal HCMV infection can adversely impact lung development, leading to pneumonia and acute lung injury. We have successfully developed a small-animal model that closely recapitulates fetal and neonatal lung development and provides a valuable, biologically relevant tool for an understanding of the lung pathogenesis of HCMV as well as other human respiratory viruses. Additionally, this model would greatly facilitate the development and testing of new antiviral therapies for HCMV along with select human pulmonary pathogens.
Asunto(s)
Infecciones por Citomegalovirus/congénito , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Alveolos Pulmonares/virología , Proteínas Asociadas a Surfactante Pulmonar/metabolismo , Replicación Viral/fisiología , Animales , Apoptosis/fisiología , Western Blotting , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones SCID , Alveolos Pulmonares/citología , Estadísticas no Paramétricas , Proteínas Virales/aislamiento & purificaciónRESUMEN
Certain ritonavir resistance mutations impair HIV infectivity through incomplete Gag processing by the mutant viral protease. Analysis of the mutant virus phenotype indicates that accumulation of capsid-spacer peptide 1 precursor protein in virus particles impairs HIV infectivity and that the protease mutant virus is arrested during the early postentry stage of HIV infection before proviral DNA synthesis. However, activation of the target cell can rescue this defect, implying that specific host factors expressed in activated cells can compensate for the defect in ritonavir-resistant HIV. This ability to rescue impaired HIV replication presented a unique opportunity to identify host factors involved in postentry HIV replication, and we designed a functional genetic screen so that expression of a given host factor extracted from activated T cells would lead directly to its discovery by rescuing mutant virus replication in nonactivated T cells. We identified the cellular heat shock protein 90 kDa α (cytosolic), class B member 1 (HSP90AB1) as a host factor that can rescue impaired replication of ritonavir-resistant HIV. Moreover, we show that pharmacologic inhibition of HSP90AB1 with 17-(allylamino)-17-demethoxygeldanamycin (tanespimycin) has potent in vitro anti-HIV activity and that ritonavir-resistant HIV is hypersensitive to the drug. These results suggest a possible role for HSP90AB1 in postentry HIV replication and may provide an attractive target for therapeutic intervention.
Asunto(s)
Farmacorresistencia Viral/fisiología , VIH/fisiología , Proteínas HSP90 de Choque Térmico/metabolismo , Provirus/fisiología , Ritonavir , Linfocitos T/virología , Replicación Viral/fisiología , Benzoquinonas/farmacología , Farmacorresistencia Viral/efectos de los fármacos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/fisiología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/genética , Humanos , Células Jurkat , Lactamas Macrocíclicas/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Linfocitos T/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Although antiretroviral therapy (ART) greatly suppresses HIV replication, lymphoid tissues remain a sanctuary site where the virus may replicate. Tracking the earliest steps of HIV spread from these cellular reservoirs after drug cessation is pivotal for elucidating how infection can be prevented. In this study, we developed an in vivo model of HIV persistence in which viral replication in the lymphoid compartments of humanized mice was inhibited by the HIV reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) to very low levels, which recapitulated ART-suppression in HIV-infected individuals. Using a combination of RNAscope in situ hybridization (ISH) and immunohistochemistry (IHC), we quantitatively investigated the distribution of HIV in the lymphoid tissues of humanized mice during active infection, EFdA suppression, and after drug cessation. The lymphoid compartments of EFdA-suppressed humanized mice harbored very rare transcription/translation-competent HIV reservoirs that enable viral rebound. Our data provided the visualization and direct measurement of the early steps of HIV reservoir expansion within anatomically intact lymphoid tissues soon after EFdA cessation and suggest a strategy to enhance therapeutic approaches aimed at eliminating the HIV reservoir.
Asunto(s)
Desoxiadenosinas/farmacología , Reservorios de Enfermedades/virología , VIH-1/fisiología , Tejido Linfoide/virología , Inhibidores de la Transcriptasa Inversa/farmacología , Replicación Viral/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Femenino , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Humanos , Ratones , Ratones TransgénicosRESUMEN
BACKGROUND: Internal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated. RESULTS: A series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences. CONCLUSION: The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.
RESUMEN
RNA and DNA aptamers specific for HIV-1 reverse transcriptase (RT) can inhibit reverse transcription in vitro. RNA aptamers have been shown to potently block HIV-1 replication in culture. We previously reported mutants of HIV-1 RT with substitutions N255D or N265D that display resistance to the DNA aptamer RT1t49. Variant viruses bearing these mutations singly or in combination were compromised for replication. In order to address the wider applicability of such aptamers, HIV-1 RT variants containing the N255D, N265D or both (Dbl) were tested for the extent of their cross-resistance to other DNA/RNA aptamers as well as to other RT inhibitors. Both N265D and Dbl RTs were resistant to most aptamers tested. N255D mutant displayed mild resistance to two of the DNA aptamers, little change in sensitivity to three and hypersensitivity to one. Although all mutants displayed wild type-like ribonuclease H activity, their activity was compromised under conditions that prevent re-binding. This suggests that the processivity defect caused by these mutations can also affect RNase H function thus contributing further to the replication defect in mutant viruses. These results indicate that mutants conferring resistance to anti-RT aptamers significantly affect many HIV-1 RT enzymatic activities, which could contribute to preventing the development of resistance in vivo. If such mutations were to arise in vivo, our results suggest that variant viruses should remain susceptible to many existing anti-RT inhibitors. This result was tempered by the observation that NRTI-resistance mutations such as K65R can confer resistance to some anti-RT aptamers.
RESUMEN
Highly potent broadly neutralizing human monoclonal antibodies hold promise for HIV prophylaxis and treatment. We used the SCID-hu Thy/Liv and BLT humanized mouse models to study the efficacy of these antibodies, primarily PG16, against HIV-1 clades A, B, and C. PG16 targets a conserved epitope in the V1/V2 region of gp120 common to 70-80% of HIV-1 isolates from multiple clades and has extremely potent in vitro activity against HIVJR-CSF. PG16 was highly efficacious in SCID-hu mice as a single intraperitoneal administration the day before inoculation of R5-tropic HIV directly into their Thy/Liv implants and demonstrated even greater efficacy if PG16 administration was continued after Thy/Liv implant HIV inoculation. However, PG16 as monotherapy had no activity in humanized mice with established R5-tropic HIV infection. These results provide evidence of tissue penetration of the antibodies, which could aid in their ability to prevent infection if virus crosses the mucosal barrier.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Anti-VIH/uso terapéutico , Infecciones por VIH/terapia , VIH-1/inmunología , Animales , Modelos Animales de Enfermedad , Proteína gp120 de Envoltorio del VIH/inmunología , Masculino , Ratones , Ratones SCID , Resultado del TratamientoRESUMEN
We previously showed that reduced infectivity of HIV with incompletely processed capsid-spacer protein 1 (CA-SP1) is rescued by cellular activation or increased expression of HSP90AB1, a member of the cytosolic heat shock protein 90 family. Here we show that HSP90AB1 is present in HIV virions and that HSP90AB1, but not nonfunctional mutated HSP90AB1(E42A+D88A), restores infectivity to HIV with mutations in CA that alter core stability. Further, the CA mutants were hypersensitive to pharmacological inhibition of HSP90AB1. In agreement with Roesch et al. (2012), we found that culturing HIV at 39.5°C enhanced viral infectivity up to 30-fold in human peripheral blood mononuclear cells (p=0.002) and rescued CA-mutant infectivity in nonactivated cells, concurrent with elevated expression of HSP90AB1 during hyperthermia. In sum, the transdominant effect of HSP90AB1 on CA-mutant HIV infectivity suggests a potential role for this class of cellular chaperones in HIV core stability and uncoating.
Asunto(s)
Proteínas de la Cápside/genética , VIH/genética , VIH/patogenicidad , Proteínas HSP90 de Choque Térmico/metabolismo , Línea Celular , Células HEK293 , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Calor , Humanos , Células Jurkat , Leucocitos Mononucleares/virología , Mutación , Linfocitos T/virología , Proteínas del Núcleo Viral/genéticaRESUMEN
Humanized Bone marrow/Liver/Thymus (BLT) mice recapitulate the mucosal transmission of HIV, permitting study of early events in HIV pathogenesis and evaluation of preexposure prophylaxis methods to inhibit HIV transmission. Human hematopoiesis is reconstituted in NOD-scid mice by implantation of human fetal liver and thymus tissue to generate human T cells plus intravenous injection of autologous liver-derived CD34(+) hematopoietic stem cells to engraft the mouse bone marrow. In side-by-side comparisons, we show that NOD-scid mice homozygous for a deletion of the IL-2Rγ-chain (NOD-scid IL-2Rγ(-/-)) are far superior to NOD-scid mice in both their peripheral blood reconstitution with multiple classes of human leukocytes (e.g., a mean of 182 versus 14 CD4(+) T cells per µl 12 weeks after CD34(+) injection) and their susceptibility to intravaginal HIV exposure (84% versus 11% viremic mice at 4 weeks). These results should speed efforts to obtain preclinical animal efficacy data for new HIV drugs and microbicides.
Asunto(s)
Infecciones por VIH/transmisión , VIH/inmunología , Subunidad gamma Común de Receptores de Interleucina/fisiología , Leucocitos/fisiología , Animales , Antígenos CD34 , Susceptibilidad a Enfermedades , Femenino , VIH/fisiología , Humanos , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/inmunología , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Trasplante Heterólogo , VaginaRESUMEN
BACKGROUND: The HIV-1 maturation inhibitor, 3-O-(3',3'-dimethylsuccinyl) betulinic acid (bevirimat, PA-457) is a promising drug candidate with 10 nM in vitro antiviral activity against multiple wild-type (WT) and drug-resistant HIV-1 isolates. Bevirimat has a novel mechanism of action, specifically inhibiting cleavage of spacer peptide 1 (SP1) from the C-terminus of capsid which results in defective core condensation. METHODS AND FINDINGS: Oral administration of bevirimat to HIV-1-infected SCID-hu Thy/Liv mice reduced viral RNA by >2 log(10) and protected immature and mature T cells from virus-mediated depletion. This activity was observed at plasma concentrations that are achievable in humans after oral dosing, and bevirimat was active up to 3 days after inoculation with both WT HIV-1 and an AZT-resistant HIV-1 clinical isolate. Consistent with its mechanism of action, bevirimat caused a dose-dependent inhibition of capsid-SP1 cleavage in HIV-1-infected human thymocytes obtained from these mice. HIV-1 NL4-3 with an alanine-to-valine substitution at the N-terminus of SP1 (SP1/A1V), which is resistant to bevirimat in vitro, was also resistant to bevirimat treatment in the mice, and SP1/AIV had replication and thymocyte kinetics similar to that of WT NL4-3 with no evidence of fitness impairment in in vivo competition assays. Interestingly, protease inhibitor-resistant HIV-1 with impaired capsid-SP1 cleavage was hypersensitive to bevirimat in vitro with a 50% inhibitory concentration 140 times lower than for WT HIV-1. CONCLUSIONS: These results support further clinical development of this first-in-class maturation inhibitor and confirm the usefulness of the SCID-hu Thy/Liv model for evaluation of in vivo antiretroviral efficacy, drug resistance, and viral fitness.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Succinatos/farmacología , Triterpenos/farmacología , Administración Oral , Animales , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/sangre , Western Blotting , Citometría de Flujo , VIH-1/fisiología , Humanos , Depleción Linfocítica , Masculino , Ratones , Ratones SCID , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Succinatos/administración & dosificación , Succinatos/sangre , Timo/virología , Triterpenos/administración & dosificación , Triterpenos/sangre , Replicación Viral/efectos de los fármacosRESUMEN
RNA molecules can be powerful inhibitors of HIV-1 replication. To determine the relative efficacy of siRNAs and RNA aptamers, a direct comparison of three anti-HIV reverse transcriptase aptamers and three shRNAs targeted to HIV-1(R3b) was made. U6 promoter-driven anti-HIV genes were delivered into CEMx174 cells via a retroviral vector, and transduced cells were sorted out via green fluorescent protein function and challenged with HIV. The results show that, at low virus input, shRNAs can block HIV as efficiently as aptamers. When expressed in target cells, both classes of inhibitors blocked early events of reverse transcription, suggesting they are both able to access intracellular reverse transcription complexes. However, at higher multiplicities of infection (m.o.i. of 50), while the aptamers could efficiently inhibit HIV replication, shRNAs did not. RNase protection assays indicated similar steady-state levels or nucleocytoplasmic distribution showing that the differential efficacy was not a reflection of intracellular concentration. The higher potency of anti-RT aptamers could be due to their ability to inhibit two successive rounds of reverse transcription owing to their unique ability to be encapsidated into virion particles. Furthermore, anti-RT aptamers expressed in T cells afforded protection against high-dose infection by chimeric RT-SHIV viruses.
Asunto(s)
Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/fisiología , ARN Viral/genética , ARN/metabolismo , Replicación Viral , Emparejamiento Base , Línea Celular , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/genética , Cinética , Regiones Promotoras Genéticas/genética , ARN/química , ARN/genética , ARN Nuclear Pequeño/genética , ARN Viral/química , ARN Viral/metabolismo , Factores de Tiempo , Transcripción Genética/genéticaRESUMEN
RNA aptamers derived by SELEX (systematic evolution of ligands by exponential enrichment) and specific for human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) bind at the template-primer cleft with high affinity and inhibit its activity. In order to determine the potential of such template analog RT inhibitors (TRTIs) to inhibit HIV-1 replication, 10 aptamers were expressed with flanking, self-cleaving ribozymes to generate aptamer RNA transcripts with minimal flanking sequences. From these, six aptamers (70.8,13, 70.15, 80.55,65, 70.28, 70.28t34, and 1.1) were selected based on binding constants (K(d)) and the degree of inhibition of RT in vitro (50% inhibitory concentration [IC(50)]). These six aptamers were each stably expressed in 293T cells followed by transfection of a molecular clone of HIV(R3B). Analysis of the virion particles revealed that the aptamers were encapsidated into the virions released and that the packaging of the viral genomic RNA or the cognate primer, tRNA(Lys)(3), was apparently unaffected. Infectivity of virions produced from 293T cell lines expressing the aptamers, as measured by infecting LuSIV reporter cells, was reduced by 90 to 99.5% compared to virions released from cells not expressing any aptamers. PCR analysis of newly made viral DNA upon infection with virions containing any of the three aptamers with the strongest binding affinities (70.8,13, 70.15, and 80.55,65) showed that all three were able to form the minus-strand strong-stop DNA. However, virions with the aptamers 70.8 and 70.15 were defective for first-strand transfer, suggesting an early block in viral reverse transcription. Jurkat T cells expressing each of the three aptamers, when infected with HIV(R3B), completely blocked the spread of HIV in culture. We found that the replication of nucleoside analog RT inhibitor-, nonnucleoside analog RT inhibitor-, and protease inhibitor-resistant viruses was strongly suppressed by the three aptamers. In addition, some of the HIV subtypes were severely inhibited (subtypes A, B, D, E, and F), while others were either moderately inhibited (subtypes C and O) or were naturally resistant to inhibition (chimeric A/D subtype). As virion-encapsidated TRTIs can predispose virions for inhibition immediately upon entry, they should prove to be efficacious agents in gene therapy approaches for AIDS.
Asunto(s)
Fármacos Anti-VIH/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , ARN/química , Inhibidores de la Transcriptasa Inversa/farmacología , Replicación Viral/efectos de los fármacos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Sitios de Unión , Línea Celular , Transcriptasa Inversa del VIH/metabolismo , VIH-1/fisiología , Células HeLa , Humanos , Células Jurkat , Ligandos , Conformación de Ácido Nucleico , ARN/metabolismo , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/metabolismo , Moldes Genéticos , Virión/fisiologíaRESUMEN
We isolated two template analog reverse transcriptase (RT) inhibitor-resistant mutants of human immunodeficiency virus (HIV) type 1 RT by using the DNA aptamer, RT1t49. The mutations associated, N255D or N265D, displayed low-level resistance to RT1t49, while high-level resistance could be observed when both mutations were present (Dbl). Molecular clones of HIV that contained the mutations produced replication-defective virions. All three RT mutants displayed severe processivity defects. Thus, while biochemical resistance to the DNA aptamer RT1t49 can be generated in vitro via multiple mutations, the overlap between the aptamer- and template-primer-binding pockets favors mutations that also affect the RT-template-primer interaction. Therefore, viruses with such mutations are replication defective. Potent inhibition and a built-in mechanism to render aptamer-resistant viruses replication defective make this an attractive class of inhibitors.
Asunto(s)
Fármacos Anti-VIH/farmacología , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , Mutación , Inhibidores de la Transcriptasa Inversa/farmacología , Replicación Viral/efectos de los fármacos , ADN Viral/metabolismo , Farmacorresistencia Viral , Infecciones por VIH/virología , VIH-1/enzimología , VIH-1/genética , VIH-1/fisiología , Humanos , Ligandos , Moldes GenéticosRESUMEN
Reverse transcriptase (RT) and integrase (IN) are two key catalytic enzymes encoded by all retroviruses. It has been shown that a specific interaction occurs between the human immunodeficiency virus type 1 (HIV-1) RT and IN proteins (X. Wu, H. Liu, H. Xiao, J. A. Conway, E. Hehl, G. V. Kalpana, V. R. Prasad, and J. C. Kappes, J. Virol. 73:2126-2135, 1999). We have now further examined this interaction to map the binding domains and to determine the effects of interaction on enzyme function. Using recombinant purified proteins, we have found that both a HIV-1 RT heterodimer (p66/p51) and its individual subunits, p51 and p66, are able to bind to HIV-1 IN. An oligomerization-defective mutant of IN, V260E, retained the ability to bind to RT, showing that IN oligomerization may not be required for interaction. Furthermore, we report that the C-terminal domain of IN, but not the N-terminal zinc-binding domain or the catalytic core domain, was able to bind to heterodimeric RT. Deletion analysis to map the IN-binding domain on RT revealed two separate IN-interacting domains: the fingers-palm domain and the carboxy-terminal half of the connection subdomain. The carboxy-terminal domain of IN alone retained its interaction with both the fingers-palm and the connection-RNase H fragments of RT, but not with the half connection-RNase H fragment. This interaction was not bridged by nucleic acids, as shown by micrococcal nuclease treatment of the proteins prior to the binding reaction. The influences of IN and RT on each other's activities were investigated by performing RT processivity and IN-mediated 3' processing and joining reactions in the presence of both proteins. Our results suggest that, while IN had no influence on RT processivity, RT stimulated the IN-mediated strand transfer reaction in a dose-dependent manner up to 155-fold. Thus, a functional interaction between these two viral enzymes may occur during viral replication.