RESUMEN
Coreceptor switching from CCR5 to CXCR4 is common during chronic HIV-1 infection, but is even more common in individuals who have failed antiretroviral therapy (ART). Prior studies have suggested rapid mutation and/or recombination of HIV-1 envelope (env) genes during coreceptor switching. We compared the functional and genotypic changes in env of viruses from viremic subjects who had failed ART just before and after coreceptor switching and compared those to viruses from matched subjects without coreceptor switching. Analysis of multiple unique functional env clones from each subject revealed extensive diversity at both sample time points and rapid diversification of sequences during the 4-month interval in viruses from both 9 subjects with coreceptor switching and 15 control subjects. Only two subjects had envs with evidence of recombination. Three findings distinguished env clones from subjects with coreceptor switching from controls: (1) lower entry efficiency via CCR5; (2) longer V1/V2 regions; and (3), lower nadir CD4 T cell counts during prior years of infection. Most of these subjects harbored virus with lower replicative capacity associated with protease (PR) and/or reverse transcriptase inhibitor resistance mutations, and the extensive diversification tended to lead either to improved entry efficiency via CCR5 or the gain of entry function via CXCR4. These results suggest that R5X4 or X4 variants emerge from a diverse, low-fitness landscape shaped by chronic infection, multiple ART resistance mutations, the availability of target cells, and reduced entry efficiency via CCR5.
Asunto(s)
Variación Genética , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/fisiología , Receptores del VIH/metabolismo , Tropismo Viral , Acoplamiento Viral , Fármacos Anti-VIH/uso terapéutico , Evolución Molecular , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Mutación , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Recombinación Genética , Viremia , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
In about half of patients infected with HIV-1 subtype B, viral populations shift from utilizing the transmembrane protein CCR5 to CXCR4, as well as or instead of CCR5, during late stage progression of the disease. How the relative adhesion efficiency and fusion competency of the viral Env proteins relate to infection during this transition is not well understood. Using a virus-cell fusion assay and live-cell single-molecule force spectroscopy, we compare the entry competency of viral clones to tensile strengths of the individual Env-receptor bonds of Env proteins obtained from a HIV-1 infected patient prior to and during coreceptor switching. The results suggest that the genetic determinants of viral entry were predominantly enriched in the C3, HR1 and CD regions rather than V3. Env proteins can better mediate entry into cells after coreceptor switch; this effective entry capacity does not correlate with the bond strengths between viral Env and cellular receptors.
Asunto(s)
VIH-1/fisiología , Acoplamiento Viral , Internalización del Virus , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Antígenos CD4/metabolismo , Línea Celular , Humanos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Receptores del VIH/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
CCR5 is the major HIV-1 entry coreceptor. RANTES/CCL5 analogs are more potent inhibitors of infection than native chemokines; one class activates and internalizes CCR5, one neither activates nor internalizes, and a third partially internalizes without activation. Here we show that mutations in CCR5 transmembrane domains differentially impact the activity of these three inhibitor classes, suggesting that the transmembrane region of CCR5, a key interaction site for inhibitors, is a sensitive molecular switch, modulating receptor activity.
Asunto(s)
Quimiocina CCL5/fisiología , Mutación , Receptores CCR5/genética , Receptores CCR5/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Fármacos Anti-VIH/farmacología , Línea Celular , Quimiocina CCL5/agonistas , Quimiocina CCL5/antagonistas & inhibidores , Quimiocina CCL5/genética , VIH-1/patogenicidad , VIH-1/fisiología , Interacciones Huésped-Patógeno , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Receptores CCR5/química , Transducción de Señal , Internalización del Virus/efectos de los fármacosRESUMEN
The great majority of human immunodeficiency virus type 1 (HIV-1) strains enter CD4+ target cells by interacting with one of two coreceptors, CCR5 or CXCR4. Here we describe a transmitted/founder (T/F) virus (ZP6248) that was profoundly impaired in its ability to utilize CCR5 and CXCR4 coreceptors on multiple CD4+ cell lines as well as primary human CD4+ T cells and macrophages in vitro yet replicated to very high titers (>80 million RNA copies/ml) in an acutely infected individual. Interestingly, the envelope (Env) glycoprotein of this clade B virus had a rare GPEK sequence in the crown of its third variable loop (V3) rather than the consensus GPGR sequence. Extensive sequencing of sequential plasma samples showed that the GPEK sequence was present in virtually all Envs, including those from the earliest time points after infection. The molecularly cloned (single) T/F virus was able to replicate, albeit poorly, in cells obtained from ccr5Δ32 homozygous donors. The ZP6248 T/F virus could also infect cell lines overexpressing the alternative coreceptors GPR15, APJ, and FPRL-1. A single mutation in the V3 crown sequence (GPEK->GPGK) of ZP6248 restored its infectivity in CCR5+ cells but reduced its ability to replicate in GPR15+ cells, indicating that the V3 crown motif played an important role in usage of this alternative coreceptor. These results suggest that the ZP6248 T/F virus established an acute in vivo infection by using coreceptor(s) other than CCR5 or CXCR4 or that the CCR5 coreceptor existed in an unusual conformation in this individual.
Asunto(s)
VIH-1/fisiología , Receptores del VIH/metabolismo , Tropismo Viral , Secuencias de Aminoácidos , Sustitución de Aminoácidos/genética , Receptores de Apelina , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Expresión Génica , Humanos , Macrófagos/virología , Receptores de Formil Péptido/genética , Receptores de Formil Péptido/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Lipoxina/genética , Receptores de Lipoxina/metabolismo , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Viral resistance to small molecule allosteric inhibitors of CCR5 is well documented, and involves either selection of preexisting CXCR4-using HIV-1 variants or envelope sequence evolution to use inhibitor-bound CCR5 for entry. Resistance to macromolecular CCR5 inhibitors has been more difficult to demonstrate, although selection of CXCR4-using variants might be expected. We have compared the in vitro selection of HIV-1 CC1/85 variants resistant to either the small molecule inhibitor maraviroc (MVC) or the macromolecular inhibitor 5P12-RANTES. High level resistance to MVC was conferred by the same envelope mutations as previously reported after 16-18 weeks of selection by increasing levels of MVC. The MVC-resistant mutants were fully sensitive to inhibition by 5P12-RANTES. By contrast, only transient and low level resistance to 5P12-RANTES was achieved in three sequential selection experiments, and each resulted in a subsequent collapse of virus replication. A fourth round of selection by 5P12-RANTES led, after 36 weeks, to a "resistant" variant that had switched from CCR5 to CXCR4 as a coreceptor. Envelope sequences diverged by 3.8% during selection of the 5P12-RANTES resistant, CXCR4-using variants, with unique and critical substitutions in the V3 region. A subset of viruses recovered from control cultures after 44 weeks of passage in the absence of inhibitors also evolved to use CXCR4, although with fewer and different envelope mutations. Control cultures contained both viruses that evolved to use CXCR4 by deleting four amino acids in V3, and others that maintained entry via CCR5. These results suggest that coreceptor switching may be the only route to resistance for compounds like 5P12-RANTES. This pathway requires more mutations and encounters more fitness obstacles than development of resistance to MVC, confirming the clinical observations that resistance to small molecule CCR5 inhibitors very rarely involves coreceptor switching.
Asunto(s)
Antagonistas de los Receptores CCR5 , Quimiocinas CC/farmacología , Evolución Molecular Dirigida , Farmacorresistencia Viral/efectos de los fármacos , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Secuencia de Aminoácidos , Ciclohexanos/farmacología , Proteínas gp160 de Envoltorio del VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Maraviroc , Datos de Secuencia Molecular , Receptores CCR5/química , Receptores CXCR4/química , Triazoles/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Coreceptor switching from CCR5 to CXCR4 is less common in subtype C HIV-1 infection than in subtype B for reasons that are unclear. We have examined sequential virus samples from a subtype C-infected child who had evidence of coreceptor switching. METHODS: To examine HIV-1 envelope evolution towards CXCR4 usage, env sequences were correlated with phenotypic characteristics determined by entry assays, as well as the ability to use alternative coreceptors such as FPRL1, CCR3, CCR8 and others. The value of a phenotype predictor based on V3 sequences was also assessed. RESULTS: Ninety-three sequences revealed 3 distinct coexistent virus lineages and only some members of one lineage evolved to use CXCR4. These lineages also had diverse alternative coreceptor patterns including the ability to use FPRL1, CCR3, CCR8, APJ, CMKLR1, RDC-1, CXCR6, CCR1, GPCR1, GPR15 and CCR6. Coreceptor switching was associated with extensive and rapid sequence divergence in the V1/V2 region in addition to V3 changes. Furthermore, interlineage recombination within the C2 region resulted in low predictability of a V3 sequence-based phenotype algorithm, and highlighted the importance of V1/V2 and V3 sequences in coreceptor usage. CONCLUSION: These results suggest that the evolution to coreceptor switching in subtype C infection requires more mutations than other subtypes, and this contributes to the reduced incidence of R5X4 viruses.
Asunto(s)
VIH-1/genética , Receptores CCR5/genética , Receptores CXCR4/genética , Niño , Clonación Molecular , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Humanos , Datos de Secuencia Molecular , Fenotipo , Filogenia , Receptores CCR5/inmunología , Receptores CXCR4/inmunología , Receptores de Formil Péptido/genética , Receptores de Formil Péptido/inmunología , Receptores de Lipoxina/genética , Receptores de Lipoxina/inmunología , Recombinación Genética/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
CC Chemokine Receptor 5 (CCR5) is an important mediator of chemotaxis and the primary coreceptor for HIV-1. A recent report by other researchers suggested that primary T cells harbor pools of intracellular CCR5. With the use of a series of complementary techniques to measure CCR5 expression (antibody labeling, Western blot, quantitative reverse transcription polymerase chain reaction), we established that intracellular pools of CCR5 do not exist and that the results obtained by the other researchers were false-positives that arose because of the generation of irrelevant binding sites for anti-CCR5 antibodies during fixation and permeabilization of cells.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Receptores CCR5/metabolismo , Western Blotting , Linfocitos T CD4-Positivos/química , Linfocitos T CD8-positivos/química , Separación Celular , Citoplasma/química , Citoplasma/metabolismo , Reacciones Falso Positivas , Citometría de Flujo , Humanos , Receptores CCR5/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fijación del TejidoRESUMEN
Resistance of human immunodeficiency virus type 1 (HIV-1) to small-molecule CCR5 inhibitors is well demonstrated, but resistance to macromolecular CCR5 inhibitors (e.g., PSC-RANTES) that act by both CCR5 internalization and receptor blockade had not been reported until recently (3). The report of a single simian-human immunodeficiency virus SHIV(SF162-p3) variant with one V3 and one gp41 sequence change in gp160 that conferred both altered replicative fitness and resistance to PSC-RANTES was therefore surprising. We introduced the same two mutations into both the parental HIV-1(SF162) and the macaque-adapted SHIV(SF162-p3) and found minor differences in entry fitness but no changes in sensitivity to inhibition by either PSC-RANTES or the small-molecule allosteric inhibitor TAK-779. We attribute the earlier finding to confounding fitness effects with inhibitor sensitivity.
Asunto(s)
Quimiocina CCL5/farmacología , Resistencia a Medicamentos/genética , VIH-1/genética , Mutación , Virus de la Inmunodeficiencia de los Simios/genética , Animales , Fármacos Anti-VIH/farmacología , Antagonistas de los Receptores CCR5 , VIH-1/patogenicidad , Humanos , Macaca , Receptores Virales/antagonistas & inhibidores , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Replicación Viral/efectos de los fármacosRESUMEN
Fitness epistasis, the interaction among alleles at different loci in their effects on fitness, has potentially important consequences for adaptive evolution. We investigated fitness epistasis among amino acids of a functionally important region of the human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein (gp120). Seven mutations putatively involved in the adaptation of the second conserved to third variable protein region (C2-V3) to the use of an alternative host-cell chemokine coreceptor (CXCR4) for cell entry were engineered singly and in combinations on the wild-type genetic background and their effects on viral infectivity were measured. Epistasis was found to be common and complex, involving not only pairwise interactions, but also higher-order interactions. Interactions could also be surprisingly strong, changing fitness by more than 9 orders of magnitude, which is explained by some single mutations being practically lethal. A consequence of the observed epistasis is that many of the minimum-length mutational trajectories between the wild type and the mutant with highest fitness on cells expressing the alternative coreceptor are selectively inaccessible. These results may help explain the difficulty of evolving viruses that use the alternative coreceptor in culture and the delayed evolution of this phenotype in natural infection. Knowledge of common, complex, and strong fitness interactions among amino acids is necessary for a full understanding of protein evolution.
Asunto(s)
Adaptación Fisiológica/genética , Epistasis Genética , Aptitud Genética , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , Secuencia de Aminoácidos , Evolución Molecular , Proteína gp120 de Envoltorio del VIH/química , Humanos , Datos de Secuencia Molecular , Mutación/genética , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismoRESUMEN
Effective strategies for preventing human immunodeficiency virus infection are urgently needed, but recent failures in key clinical trials of vaccines and microbicides highlight the need for new approaches validated in relevant animal models. Here, we show that 2 new chemokine (C-C motif) receptor 5 inhibitors, 5P12-RANTES (regulated on activation, normal T cell expressed and secreted) and 6P4-RANTES, fully protect against infection in the rhesus vaginal challenge model. These highly potent molecules, which are amenable to low-cost production, represent promising new additions to the microbicides pipeline.
Asunto(s)
Quimiocina CCL5/uso terapéutico , Quimiocinas/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Vagina/virología , Administración Tópica , Animales , Quimiocina CCL5/administración & dosificación , Quimiocinas/administración & dosificación , Quimiocinas/genética , Femenino , Macaca , ARN Viral/sangre , ARN Viral/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/uso terapéutico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carga ViralRESUMEN
PURPOSE OF REVIEW: To present recent information on the evolution of coreceptor use from CCR5 alone to CCR5 and CXCR4, the impact CCR5 inhibitors have on this process, and new insights into HIV-1 binding to CD4 and CCR5. RECENT FINDINGS: The findings that are summarized include resistance to CCR5 inhibitors, genotypic predictors of coreceptor use, the link between coreceptor use and cell tropism, and new data on CCR5 structure and function. SUMMARY: Resistance to CCR5 inhibitors is uncommon, and frequently involves selection of minor populations of R5X4 virus. Genotypic predictors of coreceptor use need to take into account the entire envelope sequence, not just V3. Genetic polymorphisms in humans that affect CCR5 or chemokines that bind CCR5 affect not only virus entry but also immune reconstitution.
Asunto(s)
Adaptación Biológica , Infecciones por VIH/virología , VIH-1/fisiología , Receptores del VIH/metabolismo , Acoplamiento Viral , Antígenos CD4/metabolismo , Farmacorresistencia Viral , Inhibidores de Fusión de VIH/farmacología , VIH-1/genética , Humanos , Polimorfismo Genético , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismoRESUMEN
New prevention strategies for use in developing countries are urgently needed to curb the worldwide HIV/AIDS epidemic. The N-terminally modified chemokine PSC-RANTES is a highly potent entry inhibitor against R5-tropic HIV-1 strains, with an inhibitory mechanism involving long-term intracellular sequestration of the HIV coreceptor, CCR5. PSC-RANTES is fully protective when applied topically in a macaque model of vaginal HIV transmission, but it has 2 potential disadvantages related to further development: the requirement for chemical synthesis adds to production costs, and its strong CCR5 agonist activity might induce local inflammation. It would thus be preferable to find a recombinant analogue that retained the high potency of PSC-RANTES but lacked its agonist activity. Using a strategy based on phage display, we set out to discover PSC-RANTES analogs that contain only natural amino acids. We sought molecules that retain the potency and inhibitory mechanism of PSC-RANTES, while trying to reduce CCR5 signaling to as low a level as possible. We identified 3 analogues, all of which exhibit in vitro potency against HIV-1 comparable to that of PSC-RANTES. The first, 6P4-RANTES, resembles PSC-RANTES in that it is a strong agonist that induces prolonged intracellular sequestration of CCR5. The second, 5P12-RANTES, has no detectable G protein-linked signaling activity and does not bring about receptor sequestration. The third, 5P14-RANTES, induces significant levels of CCR5 internalization without detectable G protein-linked signaling activity. These 3 molecules represent promising candidates for further development as topical HIV prevention strategies.
Asunto(s)
Antiinfecciosos/economía , Antiinfecciosos/farmacología , Antivirales/farmacología , Quimiocinas/farmacología , VIH/efectos de los fármacos , Ingeniería de Proteínas , Proteínas Recombinantes/farmacología , Quimiocina CCL5/química , Endocitosis/efectos de los fármacos , Células HeLa , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Receptores CCR5/metabolismo , Receptores Virales/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacosRESUMEN
The envelope gene (env) of human immunodeficiency virus type 1 (HIV-1) undergoes rapid divergence from the transmitted sequence and increasing diversification during the prolonged course of chronic infection in humans. In about half of infected individuals or more, env evolution leads to expansion of the use of entry coreceptor from CCR5 alone to CCR5 and CXCR4. The stochastic nature of this coreceptor switch is not well explained by host selective forces that should be relatively constant between infected individuals. Moreover, differences in the incidence of coreceptor switching among different HIV-1 subtypes suggest that properties of the evolving virus population drive the switch. We evaluated the functional properties of sequential env clones from a patient with evidence of coreceptor switching at 5.67 years of infection. We found an abrupt decline in the ability of viruses to use CCR5 for entry at this time, manifested by a 1- to 2-log increase in susceptibility to CCR5 inhibitors and a reduced ability to infect cell lines with low CCR5 expression. There was an abnormally rapid 5.4% divergence in env sequences from 4.10 to 5.76 years of infection, with the V3 and V4/V5 regions showing the greatest divergence and evidence of positive selection. These observations suggest that a decline in the fitness of R5 virus populations may be one driving force that permits the emergence of R5X4 variants.
Asunto(s)
Receptores CCR5/fisiología , Receptores CXCR4/fisiología , Antagonistas de los Receptores CCR5 , Humanos , Receptores CCR5/análisis , Receptores CCR5/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/fisiologíaRESUMEN
After disappointing results from all efficacy trials conducted to date, the field of microbicides research now faces substantial challenges. Poor coordination among interested parties and the choice of nonvalidated scientific targets for phase III studies have hampered progress and created mistrust about the use of microbicides as a method to prevent HIV-1 sexual transmission. Although new promising strategies are available, there will need to be serious reappraisals of how decisions are made to advance the next generations of candidates into clinical trials, and the use of appropriate animal models in this process will be critical.
Asunto(s)
Fármacos Anti-VIH/administración & dosificación , Antiinfecciosos Locales/administración & dosificación , Infecciones por VIH/prevención & control , VIH-1/efectos de los fármacos , Polímeros/administración & dosificación , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Enfermedades Vaginales/prevención & control , Administración Intravaginal , Animales , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Antiinfecciosos Locales/farmacología , Antiinfecciosos Locales/uso terapéutico , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Quimioterapia Combinada , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/transmisión , Humanos , Masculino , Cooperación del Paciente , Polielectrolitos , Polímeros/farmacología , Polímeros/uso terapéutico , Primates , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Enfermedades Vaginales/tratamiento farmacológicoAsunto(s)
Fármacos Anti-VIH/uso terapéutico , Antagonistas de los Receptores CCR5 , Ciclohexanos/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Triazoles/uso terapéutico , Fármacos Anti-VIH/farmacología , Ciclohexanos/farmacología , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Maraviroc , Pronóstico , Receptores CCR5/fisiología , Receptores CXCR4/fisiología , Triazoles/farmacologíaRESUMEN
We studied the evolution of human immunodeficiency virus type 1 (HIV-1) envelope function during the process of coreceptor switching from CCR5 to CXCR4. Site-directed mutagenesis was used to introduce most of the possible intermediate mutations in the envelope for four distinct coreceptor switch mutants, each with a unique pattern of CCR5 and CXCR4 utilization that extended from highly efficient use of both coreceptors to sole use of CXCR4. Mutated envelopes with some preservation of entry function on either CCR5- or CXCR4-expressing target cells were further characterized for their sensitivity to CCR5 or CXCR4 inhibitors, soluble CD4, and the neutralizing antibodies b12-IgG and 4E10. A subset of mutated envelopes was also studied in direct CD4 or CCR5 binding assays and in envelope-mediated fusion reactions. Coreceptor switch intermediates displayed increased sensitivity to CCR5 inhibitors (except for a few envelopes with mutations in V2 or C2) that correlated with a loss in CCR5 binding. As use of CXCR4 improved, infection mediated by the mutated envelopes became more resistant to soluble CD4 inhibition and direct binding to CD4 increased. These changes were accompanied by increasing resistance to the CXCR4 inhibitor AMD3100. Sensitivity to neutralizing antibody was more variable, although infection of CXCR4-expressing targets was generally more sensitive to neutralization by both b12-IgG and 4E10 than infection of CCR5-expressing target cells. These changes in envelope function were uniform in all four series of envelope mutations and thus were independent of the final use of CCR5 and CXCR4. Decreased CCR5 and increased CD4 binding appear to be common features of coreceptor switch intermediates.
Asunto(s)
Productos del Gen env/metabolismo , VIH-1/metabolismo , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Secuencia de Aminoácidos , Línea Celular , Evolución Molecular , Productos del Gen env/genética , VIH-1/genética , VIH-1/ultraestructura , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Receptores CCR5/genética , Receptores CXCR4/genética , Internalización del VirusRESUMEN
BACKGROUND: An effective HIV-1 vaccine or microbicide must block the transmitted virus variants that initially establish a new infection; consequently, it is critical that such viruses be isolated and characterized. OBJECTIVE: To evaluate HIV-1 envelope variants from early in infection from individuals infected heterosexually with subtype A HIV-1 for their sensitivity to antibody-mediated neutralization and to inhibitors of viral entry. METHODS: Full-length subtype A HIV-1 envelope clones from 28-75 days postinfection were used to generate pseudoviruses for infection studies. The susceptibility of these pseudoviruses to neutralization by autologous and heterologous plasma and by monoclonal antibodies was examined. The sensitivity of these pseudoviruses to PSC-RANTES and TAK-779, inhibitors of CCR5, and to soluble CD4 (sCD4) was also evaluated. RESULTS: Pseudoviruses with subtype A HIV-1 envelopes from early in infection demonstrated a broad range of neutralization sensitivities to both autologous and heterologous plasma. However, neutralization by the monoclonal antibodies b12, 2G12, 4E10 and 2F5 was generally poor; notably, none of the 14 early virus variants were neutralized by 2G12 and only one was neutralized by b12. Viruses bearing these early CCR5-using envelopes were generally sensitive to the CCR5 inhibitors PSC-RANTES and TAK-779, but they demonstrated more variable sensitivity to sCD4. CONCLUSIONS: These subtype A HIV-1 variants, representing the viruses that must be blocked by antibody-based prevention strategies, vary in their susceptibility to neutralization. A subset of these HIV-1 variants from early in infection will be useful for screening candidate vaccines and microbicides.
Asunto(s)
Anticuerpos Antivirales/inmunología , Antagonistas de los Receptores CCR5 , Infecciones por VIH/virología , VIH-1/clasificación , Proteínas del Envoltorio Viral/inmunología , Amidas/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos CD4/inmunología , Quimiocina CCL5/inmunología , Femenino , Antígenos VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/inmunología , Inhibidores de Fusión de VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Kenia , Pruebas de Neutralización , Filogenia , Compuestos de Amonio Cuaternario/inmunología , Receptores CCR5/inmunologíaRESUMEN
Previous studies of HIV-1 variants transmitted from mother-to-infant have focused primarily on computational analyses of partial envelope gene sequences, rather than analyses of functional envelope variants. There are very few examples of well-characterized functional envelope clones from mother-infant pairs, especially from envelope variants representing the most prevalent subtypes worldwide. To address this, we amplified the envelope variants present in 4 mother-infant transmission pairs, all of whom were infected with subtype A and three of whom presumably transmitted HIV-1 during the breastfeeding period. Functional envelope clones were constructed, either encoding full-length envelope sequences from the mother and baby or by making chimeric envelope clones in a common backbone sequence. The infant envelope sequences were genetically homogeneous compared to the maternal viruses, and pseudoviruses bearing these envelopes all used CCR5 as a coreceptor. The infant viruses were generally resistant to neutralization by maternal antibodies present near the time of transmission. There were no notable differences in sensitivity of the mother and infant envelope variants to neutralization by heterologous plasma or monoclonal antibodies 2G12 and b12, or to inhibition by sCD4, PSC-RANTES or TAK779. This collection of viral envelopes, which can be used for making pseudotyped viruses, may be useful for examining the efficacy of interventions to block mother-infant transmission, including sera from vaccine candidates, purified antibodies under consideration for passive immunization and viral entry inhibitors.
Asunto(s)
Lactancia Materna , Productos del Gen env/inmunología , Genes env/genética , Infecciones por VIH/virología , VIH-1/fisiología , Transmisión Vertical de Enfermedad Infecciosa , Complicaciones Infecciosas del Embarazo/virología , Anticuerpos Monoclonales , Antígenos CD4 , Clonación Molecular , Femenino , Productos del Gen env/clasificación , Productos del Gen env/genética , Variación Genética , Anticuerpos Anti-VIH/análisis , Anticuerpos Anti-VIH/sangre , Inhibidores de Fusión de VIH/análisis , Inhibidores de Fusión de VIH/sangre , Infecciones por VIH/inmunología , Infecciones por VIH/transmisión , VIH-1/clasificación , VIH-1/genética , Células HeLa , Humanos , Lactante , Recién Nacido , Leche Humana/virología , Filogenia , Embarazo , Complicaciones Infecciosas del Embarazo/inmunología , Receptores CCR5 , Receptores CXCR4 , Carga ViralRESUMEN
CCR5-using human immunodeficiency virus type 1 (HIV-1) isolates typically gain CXCR4 use via multiple mutations in V3 and often V1/V2 regions of envelope, and patterns of mutations are distinct for each isolate. Here, we report that multiple CXCR4-using variants of a parental CCR5-using HIV-1 isolate, SF162, obtained by either target cell selection or CCR5 inhibition have a common mutation pattern characterized by the same two V3 mutations and that these mutations preexisted in some of the SF162 stocks. These results imply that SF162 has a single pathway for acquiring CXCR4 use and that prolonged culture is sufficient to select for R5X4 variants.