RESUMEN
The integrase strand transfer inhibitor (INSTI) dolutegravir is commonly used in combination antiretroviral therapy regimens and retains strong potency even with primary resistance mutations to some other INSTIs. Acquisition of accessory mutations to primary mutations results in significant increases in dolutegravir resistance. Previously, we reported that addition of the secondary mutation T97A can result in rapid treatment failure in individuals with INSTI mutations at positions 140 and 148. Here, we conducted a detailed case study of one of these individuals and find that T97A-containing HIV emerged from a large replicating population from only a few (≤4) viral lineages. When combined with primary INSTI resistance mutations, T97A provides a strong selective advantage; the finding that T97A-containing variants spread by replication and recombination, and persisted for months after discontinuing dolutegravir, has important implications as dolutegravir is rolled out worldwide.
Asunto(s)
Infecciones por VIH , Inhibidores de Integrasa VIH , Integrasa de VIH , VIH-1 , Quinolonas , Farmacorresistencia Viral/genética , Infecciones por VIH/tratamiento farmacológico , Integrasa de VIH/genética , Inhibidores de Integrasa VIH/farmacología , Inhibidores de Integrasa VIH/uso terapéutico , VIH-1/genética , Compuestos Heterocíclicos con 3 Anillos/farmacología , Compuestos Heterocíclicos con 3 Anillos/uso terapéutico , Humanos , Mutación , Oxazinas , Piperazinas , Piridonas/uso terapéutico , Quinolonas/farmacología , Recombinación Genética , Terapia RecuperativaRESUMEN
Combination antiretroviral therapy (cART) controls but does not eradicate HIV infection; HIV persistence is the principal obstacle to curing infections. The proportion of defective proviruses increases during cART, but the dynamics of this process are not well understood, and a quantitative analysis of how the proviral landscape is reshaped after cART is initiated is critical to understanding how HIV persists. Here, we studied longitudinal samples from HIV infected individuals undergoing long term cART using multiplexed Droplet Digital PCR (ddPCR) approaches to quantify the proportion of deleted proviruses in lymphocytes. In most individuals undergoing cART, HIV proviruses that contain gag are lost more quickly than those that lack gag. Increases in the fraction of gag-deleted proviruses occurred only after 1-2 years of therapy, suggesting that the immune system, and/or toxicity of viral re-activation helps to gradually shape the proviral landscape. After 10-15 years on therapy, there were as many as 3.5-5 times more proviruses in which gag was deleted or highly defective than those containing intact gag. We developed a provirus-specific ddPCR approach to quantify individual clones. Investigation of a clone of cells containing a deleted HIV provirus integrated in the HORMAD2 gene revealed that the cells underwent a massive expansion shortly after cART was initiated until the clone, which was primarily in effector memory cells, dominated the population of proviruses for over 6 years. The expansion of this HIV-infected clone had substantial effects on the overall proviral population.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/genética , VIH-1/aislamiento & purificación , Leucocitos Mononucleares/virología , Provirus/aislamiento & purificación , Terapia Antirretroviral Altamente Activa , Linfocitos T CD4-Positivos/virología , Proteínas de Ciclo Celular/genética , ADN Viral/sangre , ADN Viral/genética , Virus Defectuosos/genética , Genes gag , Duplicado del Terminal Largo de VIH , VIH-1/efectos de los fármacos , Humanos , Memoria Inmunológica , Reacción en Cadena de la Polimerasa Multiplex , Provirus/genética , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/virología , Factores de Tiempo , Resultado del Tratamiento , Carga ViralRESUMEN
In HIV-infected individuals on long-term antiretroviral therapy (ART), more than 40% of the infected cells are in clones. Although most HIV proviruses present in individuals on long-term ART are defective, including those in clonally expanded cells, there is increasing evidence that clones carrying replication-competent proviruses are common in patients on long-term ART and form part of the HIV reservoir that makes it impossible to cure HIV infection with current ART alone. Given the importance of clonal expansion in HIV persistence, we determined how soon after HIV acquisition infected clones can grow large enough to be detected (clones larger than ca. 1 × 105 cells). We studied 12 individuals sampled in early HIV infection (Fiebig stage III-V/VI) and 5 who were chronically infected. The recently infected individuals were started on ART at or near the time of diagnosis. We isolated more than 6,500 independent integration sites from peripheral blood mononuclear cells before ART was initiated and after 0.5-18 years of suppressive ART. Some infected clones could be detected approximately 4 weeks after HIV infection and some of these clones persisted for years. The results help to explain how the reservoir is established early and persists for years.
Asunto(s)
Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/fisiología , Leucocitos Mononucleares/virología , Adulto , Fármacos Anti-VIH/uso terapéutico , Células Clonales/virología , Progresión de la Enfermedad , Infecciones por VIH/tratamiento farmacológico , Humanos , Provirus/fisiología , Factores de Tiempo , Carga Viral , Integración Viral , Replicación ViralRESUMEN
Although APOBEC3 cytidine deaminases A3G, A3F, A3D and A3H are packaged into virions and inhibit viral replication by inducing G-to-A hypermutation, it is not known whether they are copackaged and whether they can act additively or synergistically to inhibit HIV-1 replication. Here, we showed that APOBEC3 proteins can be copackaged by visualization of fluorescently-tagged APOBEC3 proteins using single-virion fluorescence microscopy. We further determined that viruses produced in the presence of A3G + A3F and A3G + A3H, exhibited extensive comutation of viral cDNA, as determined by the frequency of G-to-A mutations in the proviral genomes in the contexts of A3G (GG-to-AG) and A3D, A3F or A3H (GA-to-AA) edited sites. The copackaging of A3G + A3F and A3G + A3H resulted in an additive increase and a modest synergistic increase (1.8-fold) in the frequency of GA-to-AA mutations, respectively. We also identified distinct editing site trinucleotide sequence contexts for each APOBEC3 protein and used them to show that hypermutation of proviral DNAs from seven patients was induced by A3G, A3F (or A3H), A3D and A3G + A3F (or A3H). These results indicate that APOBEC3 proteins can be copackaged and can comutate the same genomes, and can cooperate to inhibit HIV replication.
Asunto(s)
Citosina Desaminasa/metabolismo , Genoma Viral , VIH-1/genética , Mutación/genética , Desaminasas APOBEC , Adulto , Línea Celular , Citidina Desaminasa , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Humanos , Masculino , Tasa de Mutación , Nucleótidos/genética , Provirus/fisiología , Análisis de Secuencia de ADN , Virión/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Reservoirs of infectious HIV-1 persist despite years of combination antiretroviral therapy and make curing HIV-1 infections a major challenge. Most of the proviral DNA resides in CD4(+)T cells. Some of these CD4(+)T cells are clonally expanded; most of the proviruses are defective. It is not known if any of the clonally expanded cells carry replication-competent proviruses. We report that a highly expanded CD4(+) T-cell clone contains an intact provirus. The highly expanded clone produced infectious virus that was detected as persistent plasma viremia during cART in an HIV-1-infected patient who had squamous cell cancer. Cells containing the intact provirus were widely distributed and significantly enriched in cancer metastases. These results show that clonally expanded CD4(+)T cells can be a reservoir of infectious HIV-1.
Asunto(s)
Linfocitos T CD4-Positivos/virología , VIH-1/fisiología , Replicación Viral , Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/sangre , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/patogenicidad , Humanos , Datos de Secuencia Molecular , VirulenciaRESUMEN
Human APOBEC3A (A3A) is a single-domain cytidine deaminase that converts deoxycytidine residues to deoxyuridine in single-stranded DNA (ssDNA). It inhibits a wide range of viruses and endogenous retroelements such as LINE-1, but it can also edit genomic DNA, which may play a role in carcinogenesis. Here, we extend our recent findings on the NMR structure of A3A and report structural, biochemical and cell-based mutagenesis studies to further characterize A3A's deaminase and nucleic acid binding activities. We find that A3A binds ssRNA, but the RNA and DNA binding interfaces differ and no deamination of ssRNA is detected. Surprisingly, with only one exception (G105A), alanine substitution mutants with changes in residues affected by specific ssDNA binding retain deaminase activity. Furthermore, A3A binds and deaminates ssDNA in a length-dependent manner. Using catalytically active and inactive A3A mutants, we show that the determinants of A3A deaminase activity and anti-LINE-1 activity are not the same. Finally, we demonstrate A3A's potential to mutate genomic DNA during transient strand separation and show that this process could be counteracted by ssDNA binding proteins. Taken together, our studies provide new insights into the molecular properties of A3A and its role in multiple cellular and antiviral functions.
Asunto(s)
Citidina Desaminasa/química , Proteínas/química , Secuencia de Aminoácidos , Aminoácidos/química , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Desaminación , Proteínas de Escherichia coli/metabolismo , Transcriptasa Inversa del VIH/metabolismo , Humanos , Elementos de Nucleótido Esparcido Largo , Datos de Secuencia Molecular , Mutación , Unión Proteica , Conformación Proteica , Proteínas/genética , Proteínas/metabolismo , ARN/química , ARN/metabolismo , Alineación de Secuencia , Transcripción GenéticaRESUMEN
Tetherin is part of the cellular innate immunity and impedes cell-free transmission of viruses that bud from the plasma membrane by retaining them on the cell surface. Some viruses have evolved activities in different proteins such as Vpu (HIV-1), K-protein (KSHV), Nef (SIV) or Env (HIV-2) to downregulate tetherin and overcome its restriction. We found that chronically HTLV-1 infected T-cell lines express eightfold more tetherin than uninfected transformed T-cell lines suggesting that tetherin expression is not inhibited by the virus. We observed that even small amounts of exogenous tetherin caused the retention of HTLV-1 on the cell surface and severely reduced cell-free infectivity of HTLV-1, but that cell-cell transmission, which is more relevant for HTLV-1, was significantly less decreased. However, knock-down of tetherin expresssion resulted in a slight increase in cell-cell infection indicating that the protein does not enhance this route of transmission.
Asunto(s)
Antígenos CD/metabolismo , Linfocitos T CD4-Positivos/virología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Linfocitos T/virología , Liberación del Virus , Antígenos CD/genética , Linfocitos T CD4-Positivos/inmunología , Comunicación Celular , Línea Celular , Línea Celular Transformada , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Virus Linfotrópico T Tipo 1 Humano/crecimiento & desarrollo , Humanos , Inmunidad Innata , Interferencia de ARN , ARN Interferente Pequeño , Linfocitos T/inmunologíaRESUMEN
APOBEC3 proteins are cytidine deaminases which help defend cells against retroviral infections. One antiviral mechanism involves deaminating dC residues in minus-strand DNA during reverse transcription, resulting in G-to-A mutations in the coding strand. We investigated the effects of mouse APOBEC3 (mA3) and human APOBEC3G (hA3G) upon Moloney murine leukemia virus (MLV). We find that mA3 inactivates MLV but is significantly less effective against MLV than is hA3G. In contrast, mA3 is as potent against human immunodeficiency virus type 1 (HIV-1, lacking the protective Vif protein) as is hA3G. The two APOBEC3 proteins are packaged to similar extents in MLV particles. Dose-response profiles imply that a single APOBEC3 molecule (or oligomer) is sufficient to inactivate an MLV particle. The inactivation of MLV by mA3 and hA3G is accompanied by relatively small reductions in the amount of viral DNA in infected cells. Although hA3G induces significant levels of G-to-A mutations in both MLV and HIV DNAs, and mA3 induces these mutations in HIV DNA, no such mutations were detected in DNA synthesized by MLV inactivated by mA3. Thus, MLV has apparently evolved to partially resist the antiviral effects of mA3 and to totally resist the ability of mA3 to induce G-to-A mutation in viral DNA. Unlike the resistance of HIV-1 and human T-cell leukemia virus type 1 to hA3G, the resistance of MLV to mA3 is not mediated by the exclusion of APOBEC from the virus particle. The nature of its resistance and the mechanism of inactivation of MLV by mA3 are completely unknown.
Asunto(s)
Citidina Desaminasa/metabolismo , Citosina Desaminasa/metabolismo , ADN Viral/genética , Virus de la Leucemia Murina de Moloney/metabolismo , Inactivación de Virus , Desaminasas APOBEC , Animales , Secuencia de Bases , Línea Celular , Citidina Desaminasa/genética , Citosina Desaminasa/genética , Cartilla de ADN/genética , Humanos , Immunoblotting , Ratones , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney/genética , Mutación/genética , Análisis de Secuencia de ADNRESUMEN
Human T cell leukemia virus type 1 (HTLV-1) has evolved a remarkable strategy to thwart the antiviral effects of the cellular cytidine deaminase APOBEC3G (hA3G). HTLV-1 infects T lymphocytes in vivo, where, like HIV-1, it is likely to encounter hA3G. HIV-1 counteracts the innate antiviral activity of hA3G by producing an accessory protein, Vif, which hastens the degradation of hA3G. In contrast, HTLV-1 does not encode a Vif homologue; instead, HTLV-1 has evolved a cis-acting mechanism to prevent hA3G restriction. We demonstrate here that a peptide motif in the C terminus of the HTLV-1 nucleocapsid (NC) domain inhibits hA3G packaging into nascent virions. Mutation of amino acids within this region resulted in increased levels of hA3G incorporation into virions and increased susceptibility to hA3G restriction. Elements within the C-terminal extension of the NC domain are highly conserved among the primate T cell leukemia viruses, but this extension is absent in all other retroviral NC proteins.
Asunto(s)
Virus Linfotrópico T Tipo 1 Humano/efectos de los fármacos , Virus Linfotrópico T Tipo 1 Humano/fisiología , Nucleocápside/química , Nucleocápside/metabolismo , Nucleósido Desaminasas/farmacología , Proteínas Represoras/farmacología , Desaminasa APOBEC-3G , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia Conservada , Citidina Desaminasa , Productos del Gen gag/química , Productos del Gen gag/metabolismo , Productos del Gen vif/química , Productos del Gen vif/metabolismo , VIH-1 , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Nucleocápside/efectos de los fármacos , Péptidos/química , Estructura Terciaria de Proteína/efectos de los fármacos , Ensamble de Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Productos del Gen vif del Virus de la Inmunodeficiencia HumanaRESUMEN
It is well established that cell-free infection with human T-cell leukemia virus type 1 (HTLV-1) is less efficient than that with other retroviruses, though the specific infectivities of only a limited number of HTLV-1 isolates have been quantified. Earlier work indicated that a post-entry step in the infectious cycle accounted for the poor cell-free infectivity of HTLV-1. To determine whether variations in the pol gene sequence correlated with virus infectivity, we sequenced and phenotypically tested pol genes from a variety of HTLV-1 isolates derived from primary sources, transformed cell lines, and molecular clones. The pol genes and deduced amino acid sequences from 23 proviruses were sequenced and compared with 14 previously published sequences, revealing a limited number of amino acid variations among isolates. The variations appeared to be randomly dispersed among primary isolates and proviruses from cell lines and molecular clones. In addition, there was no correlation between reverse transcriptase sequence and the disease phenotype of the original source of the virus isolate. HTLV-1 pol gene fragments encoding reverse transcriptase were amplified from a variety of isolates and were subcloned into HTLV-1 vectors for both single-cycle infection and spreading-infection assays. Vectors carrying pol genes that matched the consensus sequence had the highest titers, and those with the largest number of variations from the consensus had the lowest titers. The molecular clone from CS-1 cells had four amino acid differences from the consensus sequence and yielded infectious titers that were approximately eight times lower than those of vectors encoding a consensus reverse transcriptase.
Asunto(s)
Variación Genética , Infecciones por HTLV-I/virología , Virus Linfotrópico T Tipo 1 Humano/enzimología , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Fenotipo , ADN Polimerasa Dirigida por ARN/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Análisis por Conglomerados , Genes pol/genética , Vectores Genéticos , Genotipo , Infecciones por HTLV-I/enzimología , Humanos , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Linfocitos T/virología , Replicación Viral/genéticaRESUMEN
Retrovirus infection proceeds by attachment of the envelope glycoprotein to a cell surface receptor, followed by fusion of the viral and cellular membranes. Once in the cell, the viral enzymes and structural proteins form a replication complex that converts the single-stranded viral genomic RNA into a double-stranded DNA, which is then integrated into the host cell chromosome. For HTLV-1, these events are not well characterized. We have developed cell culture systems, infectious molecular clones, and viral vectors that can be used to characterize the mechanisms of HTLV-1 infection and replication. Infection with cell-free HTLV-1 virions is orders of magnitude less efficient compared with other retroviruses. This inefficiency is the result of a block in the replication process after the virion is bound to the cell surface. We are determining whether this block is conferred by the viral replication enzymes, results from the actions of cellular restriction factors, reflects the need for cell-cell contact, or is caused by a combination of these factors.
Asunto(s)
Infecciones por HTLV-I/transmisión , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Replicación Viral , Animales , Antivirales/farmacología , Regulación Viral de la Expresión Génica , Humanos , Leucemia de Células T/virología , ADN Polimerasa Dirigida por ARN/metabolismoRESUMEN
A single-cycle infection assay with recombinant viral vectors was developed to study human T cell leukemia virus type I (HTLV-I) replication and its inhibition by antiviral agents. The susceptibility of HTLV-I to 6 nucleoside reverse-transcriptase inhibitors was examined. HTLV-I replication was inhibited by tenofovir, abacavir, lamivudine, zalcitabine, stavudine, and zidovudine.
Asunto(s)
Adenina/análogos & derivados , Virus Linfotrópico T Tipo 1 Humano/efectos de los fármacos , Organofosfonatos , Inhibidores de la Transcriptasa Inversa/farmacología , Adenina/farmacología , Línea Celular , Didesoxinucleósidos/farmacología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Humanos , Lamivudine/farmacología , Pruebas de Sensibilidad Microbiana , Compuestos Organofosforados/farmacología , Estavudina/farmacología , Tenofovir , Replicación Viral/efectos de los fármacos , Zalcitabina/farmacología , Zidovudina/farmacologíaRESUMEN
The genomic RNA of human T-cell leukemia virus type 1 encodes three polyproteins, Gag, Gag-Pro, and Gag-Pro-Pol, which are translated as a result of no, one, and two frameshifts, respectively. In this report we demonstrate that the 77 residues encoded at the C terminus of the Gag-Pro precursor can be collectively detected as an 8-kDa transframe protein (TFP) in virions. Mutant viruses with a C-terminally truncated TFP (19, 32, or 50 residues) had essentially a wild-type phenotype in vitro. However, a virus mutant that encoded only the Gag and Gag-Pro-Pol polyproteins due to a mutation in the second frameshift site, and hence did not produce TFP, was noninfectious. Mutation analysis of the proteolytic cleavage site between PR and TFP revealed the presence of an additional site and the existence of a p1 peptide separating protease and TFP. While removal of the cleavage site at the PR-p1 junction had a modest effect on virus replication, mutation of the p1-TFP cleavage site led to noninfectious virus and the loss of reverse transcriptase activity. Determination of the amino-terminal sequence of a hemagglutinin-tagged RT demonstrated that the same site is used in processing the Gag-Pro-Pol precursor and thus defines the start of mature RT. Neither mutation alone or in combination caused changes in the amounts or processing patterns of the Gag polyprotein, indicating that protease is active independent of its C terminus.