RESUMEN
Throughout the HIV-1 replication cycle, complex host-pathogen interactions take place in the infected cell, leading to the production of new virions. The virus modulates the host cellular machinery in order to support its life cycle, while counteracting intracellular defense mechanisms. We investigated the dynamic host response to HIV-1 infection by systematically measuring transcriptomic, proteomic, and phosphoproteomic expression changes in infected and uninfected SupT1 CD4+ T cells at five time points of the viral replication process. By means of a Gaussian mixed-effects model implemented in the new R/Bioconductor package TMixClust, we clustered host genes based on their temporal expression patterns. We identified a proteo-transcriptomic gene expression signature of 388 host genes specific for HIV-1 replication. Comprehensive functional analyses of these genes confirmed the previously described roles of some of the genes and revealed novel key virus-host interactions affecting multiple molecular processes within the host cell, including signal transduction, metabolism, cell cycle, and immune system. The results of our analysis are accessible through a freely available, dedicated and user-friendly R/Shiny application, called PEACHi2.0. This resource constitutes a catalogue of dynamic host responses to HIV-1 infection that provides a basis for a more comprehensive understanding of virus-host interactions.
Asunto(s)
Infecciones por VIH/genética , VIH-1/genética , Interacciones Huésped-Patógeno/genética , Linfocitos T CD4-Positivos/metabolismo , Perfilación de la Expresión Génica/métodos , Infecciones por VIH/virología , VIH-1/metabolismo , VIH-1/patogenicidad , Humanos , Proteoma/genética , Proteómica/métodos , Transducción de Señal , Transcriptoma/genética , Latencia del Virus/genética , Replicación Viral/genéticaRESUMEN
Despite effective treatment, HIV can persist in latent reservoirs, which represent a major obstacle toward HIV eradication. Targeting and reactivating latent cells is challenging due to the heterogeneous nature of HIV-infected cells. Here, we used a primary model of HIV latency and single-cell RNA sequencing to characterize transcriptional heterogeneity during HIV latency and reactivation. Our analysis identified transcriptional programs leading to successful reactivation of HIV expression.
Asunto(s)
Regulación Viral de la Expresión Génica/fisiología , Infecciones por VIH/metabolismo , VIH-1/fisiología , Análisis de Secuencia de ARN , Activación Viral/fisiología , Latencia del Virus/fisiología , Femenino , Infecciones por VIH/genética , Humanos , MasculinoRESUMEN
Cellular permissiveness to HIV infection is highly heterogeneous across individuals. Heterogeneity is also found across CD4+ T cells from the same individual, where only a fraction of cells gets infected. To explore the basis of permissiveness, we performed single-cell RNA-seq analysis of non-infected CD4+ T cells from high and low permissive individuals. Transcriptional heterogeneity translated in a continuum of cell states, driven by T-cell receptor-mediated cell activation and was strongly linked to permissiveness. Proteins expressed at the cell surface and displaying the highest correlation with T cell activation were tested as biomarkers of cellular permissiveness to HIV. FACS sorting using antibodies against several biomarkers of permissiveness led to an increase of HIV cellular infection rates. Top candidate biomarkers included CD25, a canonical activation marker. The combination of CD25 high expression with other candidate biomarkers led to the identification of CD298, CD63 and CD317 as the best biomarkers for permissiveness. CD25highCD298highCD63highCD317high cell population showed an enrichment of HIV-infection of up to 28 fold as compared to the unsorted cell population. The purified hyper-permissive cell subpopulation was characterized by a downregulation of interferon-induced genes and several known restriction factors. Single-cell RNA-seq analysis coupled with functional characterization of cell biomarkers provides signatures of the "HIV-permissive cell".
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Activación de Linfocitos/inmunología , Células Cultivadas , Humanos , Análisis de la Célula Individual/métodos , Replicación Viral/fisiologíaRESUMEN
Treatment of HIV-infected patients with IFN-α results in significant, but clinically insufficient, reductions of viremia. IFN induces the expression of several antiviral proteins including BST-2, which inhibits HIV by multiple mechanisms. The viral protein Vpu counteracts different effects of BST-2. We thus asked if Vpu proteins from IFN-treated patients displayed improved anti-BST-2 activities as compared to Vpu from baseline. Deep-sequencing analyses revealed that in five of seven patients treated by IFN-α for a concomitant HCV infection in the absence of antiretroviral drugs, the dominant Vpu sequences differed before and during treatment. In three patients, vpu alleles that emerged during treatment improved virus replication in the presence of IFN-α, and two of them conferred improved virus budding from cells expressing BST-2. Differences were observed for the ability to down-regulate CD4, while all Vpu variants potently down-modulated BST-2 from the cell surface. This report discloses relevant consequences of IFN-treatment on HIV properties.
Asunto(s)
Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/genética , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Interferón-alfa/uso terapéutico , Proteínas Reguladoras y Accesorias Virales/genética , Alelos , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos CD4/genética , Antígenos CD4/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , VIH-1/clasificación , VIH-1/aislamiento & purificación , VIH-1/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Fenotipo , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
Single-cell sequencing (SCS) has emerged as a valuable tool to study cellular heterogeneity in diverse fields, including virology. By studying the viral and cellular genome and/or transcriptome, the dynamics of viral infection can be investigated at single cell level. Most studies have explored the impact of cell-to-cell variation on the viral life cycle from the point of view of the virus, by analyzing viral sequences, and from the point of view of the cell, mainly by analyzing the cellular host transcriptome. In this review, we will focus on recent studies that use single-cell sequencing to explore viral diversity and cell variability in response to viral replication.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Patógeno/genética , Análisis de la Célula Individual , Virosis/genética , Virosis/virología , Fenómenos Fisiológicos de los Virus , Animales , Biología Computacional/métodos , Epigénesis Genética , Perfilación de la Expresión Génica , Genómica/métodos , Humanos , Análisis de la Célula Individual/métodosRESUMEN
Single-cell sequencing technologies, i.e., single cell analysis followed by deep sequencing investigate cellular heterogeneity in many biological settings. It was only in the past year that single-cell sequencing analyses has been applied in the field of virology, providing new ways to explore viral diversity and cell response to viral infection, which are summarized in the present review.
Asunto(s)
Genómica/métodos , Interacciones Huésped-Patógeno , Análisis de la Célula Individual/métodos , Virología/métodos , Virus/inmunología , Virus/patogenicidad , HumanosRESUMEN
Type-I interferons (IFNs) induce the expression of hundreds of cellular genes, some of which have direct antiviral activities. Although IFNs restrict different steps of HIV replication cycle, their dominant antiviral effect remains unclear. We first quantified the inhibition of HIV replication by IFN in tissue culture, using viruses with different tropism and growth kinetics. By combining experimental and mathematical analyses, we determined quantitative estimates for key parameters of HIV replication and inhibition, and demonstrate that IFN mainly inhibits de novo infection (33% and 47% for a X4- and a R5-strain, respectively), rather than virus production (15% and 6% for the X4 and R5 strains, respectively). This finding is in agreement with patient-derived data analyses.
Asunto(s)
Antivirales/farmacología , VIH-1/fisiología , Replicación Viral/efectos de los fármacos , Técnicas de Cultivo de Célula , Células HEK293 , VIH-1/efectos de los fármacos , Humanos , Interferón Tipo I/farmacología , Modelos BiológicosRESUMEN
OBJECTIVES: HIV resistance to the integrase inhibitor raltegravir in treated patients is characterized by distinct resistance pathways. We hypothesize that differences in the in vivo dynamics of HIV resistance to raltegravir are due to the genetic context of the integrase present at baseline. PATIENTS AND METHODS: We studied four patients whose viruses evolved towards different resistance pathways. The integrase baseline sequences were inserted into a reference clone. Primary resistance mutations were then introduced and their impact on viral replication capacity (RC) and resistance was measured. RESULTS: Patients A and B experienced emergence and persistence of mutation N155H under raltegravir therapy. In the integrase sequence from Patient A, N155H conferred potent resistance coupled with a lower impact on RC than Q148H. In Patient B, instead, selection of N155H could be explained by the dramatic loss of RC induced by the alternative Q148H mutation. In Patient C, N155H initially emerged and was later replaced by Q148H. In this integrase context, N155H resulted in higher RC but lower resistance than Q148H. In Patient D, Q148H rapidly emerged without appearance of N155H. This was the only patient for whom Q148H conferred higher RC and resistance than N155H. CONCLUSIONS: The emergence of different resistance mutations in patients was in full agreement with the impact of mutations in different baseline integrase contexts. Evolution towards different resistance genotypes is thus largely determined by the capacity of different integrase sequences present at baseline to minimize the effect of mutations on virus RC while allowing expression of resistance.
Asunto(s)
Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral , Integrasa de VIH/biosíntesis , Integrasa de VIH/genética , VIH/efectos de los fármacos , Mutación Missense , Pirrolidinonas/farmacología , VIH/enzimología , VIH/fisiología , Humanos , Raltegravir Potásico , Replicación Viral/efectos de los fármacosRESUMEN
The human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein plays an important role during the early stages of the retroviral life cycle and therefore is an attractive target for therapeutic intervention. We immunized rabbits with HIV-1 IN protein and developed a combinatorial single-chain variable fragment (scFv) library against IN. Five different scFv antibodies with high binding activity and specificity for IN were identified. These scFvs recognize the catalytic and C-terminal domains of IN and block the strand-transfer process. Cells expressing anti-IN-scFvs were highly resistant to HIV-1 replication due to an inhibition of the integration process itself. These results provide proof-of-concept that rabbit anti-IN-scFv intrabodies can be designed to block the early stages of HIV-1 replication without causing cellular toxicity. Therefore, these anti-IN-scFvs may be useful agents for "intracellular immunization"-based gene therapy strategies. Furthermore, because of their epitope binding characteristics, these scFvs can be used also as new tools to study the structure and function of HIV-1 IN protein.
Asunto(s)
Dominio Catalítico , Integrasa de VIH/química , Integrasa de VIH/inmunología , VIH-1/fisiología , Proteínas Recombinantes/inmunología , Anticuerpos de Cadena Única/inmunología , Replicación Viral/inmunología , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Línea Celular , Núcleo Celular/metabolismo , Mapeo Epitopo , VIH-1/inmunología , Humanos , Datos de Secuencia Molecular , Unión Proteica , Conejos , Proteínas Recombinantes/química , Anticuerpos de Cadena Única/química , Integración Viral/inmunologíaRESUMEN
HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA) library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies.
Asunto(s)
VIH-1/fisiología , Monoéster Fosfórico Hidrolasas/genética , Fosfotransferasas/genética , ARN Interferente Pequeño/genética , Western Blotting , Línea Celular , Supervivencia Celular , Biblioteca de Genes , VIH-1/genética , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Células Jurkat , Leucemia de Células T/genética , Leucemia de Células T/patología , Leucemia de Células T/virología , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas/metabolismo , Interferencia de ARN , Replicación Viral/genética , Replicación Viral/fisiología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
HIV-1 Vif protein protects viral replication in non-permissive cells by inducing degradation of APOBEC3G via ubiquitination and proteasomal pathway, although new studies indicate a putative role in Vif's direct inhibition of APOBEC3G. APOBEC3G is member of a homologous family of proteins with cytidine deaminase activity expressed with characteristic tissue specificity, that in humans consist of APOBEC1, APOBEC2, APOBEC3A-H, APOBEC4 and the activation-induced deaminase (AID), a B lymphoid protein necessary for somatic hypermutation, gene conversion and class switch recombination. In this work we show that Vif can counteract AID's activity in E. coli in absence of specific eukaryotic co-factors necessary for AID induced somatic hypermutation, gene conversion and to stimulate class switch recombination in B-cells. We show that AID inhibition is mediated by a direct protein-protein interaction via unique amino acid D118 an homologous mutant responsible for the species-specific restriction of HIV-1 Vif protein existent for APOBEC3G. These results raise the hypothesis that Vif related proteins can act as a broad inhibitor of deaminase activity. Moreover as AID and Vif evolved in different cellular environments, these results may indicate that Vif related proteins might mimic cellular factors that interact with a structural conserved domain of cytidine deaminases during evolution.