RESUMEN
APOBEC3G (A3G) restricts HIV-1 replication primarily by reducing viral cDNA and inducing G-to-A hypermutations in viral cDNA. HIV-1 encodes virion infectivity factor (Vif) to counteract A3G primarily by excluding A3G viral encapsidation. Even though the Vif-induced exclusion is robust, studies suggest that A3G is still detectable in the virion. The impact of encapsidated A3G in the HIV-1 replication is unclear. Using a highly sensitive next-generation sequencing (NGS)-based G-to-A hypermutation detecting assay, we found that wild-type HIV-1 produced from A3G-expressing T-cells induced higher G-to-A hypermutation frequency in viral cDNA than HIV-1 from non-A3G-expressing T-cells. Interestingly, although the virus produced from A3G-expressing T-cells induced higher hypermutation frequency, there was no significant difference in viral infectivity, revealing a disassociation of cDNA G-to-A hypermutation to viral infectivity. We also measured G-to-A hypermutation in the viral RNA genome. Surprisingly, our data showed that hypermutation frequency in the viral RNA genome was significantly lower than in the integrated DNA, suggesting a mechanism exists to preferentially select intact genomic RNA for viral packing. This study revealed a new insight into the mechanism of HIV-1 counteracting A3G antiviral function and might lay a foundation for new antiviral strategies.
Asunto(s)
Desaminasa APOBEC-3G , ADN Complementario , VIH-1 , Mutación , Humanos , Desaminasa APOBEC-3G/genética , Desaminasa APOBEC-3G/metabolismo , ADN Complementario/genética , ADN Viral/genética , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Infecciones por VIH/virología , VIH-1/genética , VIH-1/patogenicidad , Linfocitos T/virología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Replicación Viral/genéticaRESUMEN
Medroxyprogesterone acetate (MPA) is one of the most widely used contraceptives in the world. Epidemiologic studies have uncovered a possible link between the use of MPA and an increased risk of HIV-1 transmission. However, the understanding of the mechanism is still limited. Our previous publication demonstrated that the lysosomal activity in human vaginal epithelial cells attenuated the trafficking of viral particles during HIV-1 transcytosis. In this study, we show that treating human primary cervical epithelial cells with MPA led to a reduction in lysosomal activity. This reduction caused an increase in the intracellular HIV-1 accumulation and, consequently, an increase in viral release. Our study uncovers a novel mechanism by which MPA enhances HIV-1 release in primary cervical epithelial cells, thus providing vital information for HIV intervention and prevention.
RESUMEN
In the developing pancreas, transient Neurog3-expressing progenitors give rise to four major islet cell types: α, ß, δ, and γ; when and how the Neurog3+ cells choose cell fate is unknown. Using single-cell RNA-seq, trajectory analysis, and combinatorial lineage tracing, we showed here that the Neurog3+ cells co-expressing Myt1 (i.e., Myt1+Neurog3+) were biased toward ß cell fate, while those not simultaneously expressing Myt1 (Myt1-Neurog3+) favored α fate. Myt1 manipulation only marginally affected α versus ß cell specification, suggesting Myt1 as a marker but not determinant for islet-cell-type specification. The Myt1+Neurog3+ cells displayed higher Dnmt1 expression and enhancer methylation at Arx, an α-fate-promoting gene. Inhibiting Dnmts in pancreatic progenitors promoted α cell specification, while Dnmt1 overexpression or Arx enhancer hypermethylation favored ß cell production. Moreover, the pancreatic progenitors contained distinct Arx enhancer methylation states without transcriptionally definable sub-populations, a phenotype independent of Neurog3 activity. These data suggest that Neurog3-independent methylation on fate-determining gene enhancers specifies distinct endocrine-cell programs.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Islotes Pancreáticos/citología , Proteínas del Tejido Nervioso/metabolismo , Organogénesis/fisiología , Páncreas/metabolismo , Animales , Diferenciación Celular/genética , Linaje de la Célula/fisiología , Células Endocrinas/metabolismo , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/metabolismo , Ratones , Factores de Transcripción/metabolismoRESUMEN
Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (A3G) is a host restriction factor that impedes HIV-1 replication. Viral integrity is salvaged by HIV-1 virion infectivity factor (Vif), which mediates A3G polyubiquitination and subsequent cellular depletion. Previous studies have implied that A3G polyubiquitination is essential for Vif-induced degradation. However, the contribution of polyubiquitination to the rate of A3G degradation remains unclear. Here, we show that A3G polyubiquitination is essential for degradation. Inhibition of ubiquitin-activating enzyme E1 by PYR-41 or blocking the formation of ubiquitin chains by over-expressing the lysine to arginine mutation of ubiquitin K48 (K48R) inhibited A3G degradation. Our A3G mutagenesis study showed that lysine residues 297, 301, 303, and 334 were not sufficient to render lysine-free A3G sensitive to Vif-mediated degradation. Our data also confirm that Vif could induce ubiquitin chain formation on lysine residues interspersed throughout A3G. Notably, A3G degradation relied on the lysine residues involved in polyubiquitination. Although A3G and the A3G C-terminal mutant interacted with Vif and were modified by ubiquitin chains, the latter remained more resistant to Vif-induced degradation. Furthermore, the A3G C-terminal mutant, but not the N-terminal mutant, maintained potent antiviral activity in the presence of Vif. Taken together, our results suggest that the location of A3G ubiquitin modification is a determinant for Vif-mediated degradation, implying that in addition to polyubiquitination, other factors may play a key role in the rate of A3G degradation.
Asunto(s)
Desaminasa APOBEC-3G/metabolismo , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitina/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , ProteolisisRESUMEN
GB virus type C (GBV-C) glycoprotein E2 protein disrupts HIV-1 assembly and release by inhibiting Gag plasma membrane targeting, however the mechanism by which the GBV-C E2 inhibits Gag trafficking remains unclear. In the present study, we identified ADP-ribosylation factor 1 (ARF1) contributed to the inhibitory effect of GBV-C E2 on HIV-1 Gag membrane targeting. Expression of GBV-C E2 decreased ARF1 expression in a proteasomal degradation-dependent manner. The restoration of ARF1 expression rescued the HIV-1 Gag processing and membrane targeting defect imposed by GBV-C E2. In addition, GBV-C E2 expression also altered Golgi morphology and suppressed protein traffic through the secretory pathway, which are all consistent with a phenotype of disrupting the function of ARF1 protein. Thus, our results indicate that GBV-C E2 inhibits HIV-1 assembly and release by decreasing ARF1, and may provide insights regarding GBV-C E2's potential for a new therapeutic approach for treating HIV-1.
Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , VIH-1/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Western Blotting , Coinfección/virología , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente , Células HEK293 , Células HeLa , Humanos , Microscopía Confocal , Reacción en Cadena en Tiempo Real de la Polimerasa , Transfección , Ensamble de Virus/fisiología , Liberación del Virus/fisiologíaRESUMEN
BACKGROUND: HIV-1 viral infectivity factor (Vif) is an essential accessory protein for HIV-1 replication. The predominant function of Vif is to counteract Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G, A3G), a potent host restriction factor that inhibits HIV-1 replication. Vif mediates the proteasomal degradation of A3G and inhibits A3G translation, thus diminishing the pool of A3G that is available to be packaged into budding virion. Although Vif is robust in degrading A3G, the protection provided against A3G is not absolute. Clinical and laboratory evidence have shown that A3G is not completely excluded from HIV-1 viral particles during HIV-1 replication. It remains unclear why the viral samples are still infectious when A3G has been packaged into the virions. RESULTS: In this study, we provide evidence that Vif continues to protect HIV-1 from the deleterious effects of A3G, even after packaging of A3G has occurred. When equal amounts of A3G were packaged into budding virions, the virus expressing functional Vif was more infectious and incurred fewer G to A hypermutations in the second round of infection compared to Vif-deficient virus. A Vif mutant with a defect in viral packaging showed a reduced ability to protect the HIV-1 genome from G to A hypermutations. CONCLUSION: Our data suggest that even packaged A3G is still under the tyranny of Vif. Our work brings to light an additional caveat for any therapy that hopes to exploit the Vif-A3G axis. The ideal strategy would not only enhance A3G viral packaging, but also reduce HIV-1 Vif viral encapsidation.
Asunto(s)
Citidina Desaminasa/antagonistas & inhibidores , VIH-1/fisiología , Virión/fisiología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Desaminasa APOBEC-3G , Línea Celular , VIH-1/genética , Humanos , Mutación Puntual , ARN Viral/genética , ARN Viral/metabolismo , Ensamble de VirusRESUMEN
Although most human immunodeficiency virus type 1 (HIV-1) cases worldwide are transmitted through mucosal surfaces, transmission through the oral mucosal surface is a rare event. More than 700 bacterial species have been detected in the oral cavity. Despite great efforts to discover oral inhibitors of HIV, little information is available concerning the anti-HIV activity of oral bacterial components. Here we show that a molecule from an oral commensal bacterium, Streptococcus cristatus CC5A can induce expression of APOBEC3G (A3G) and APOBEC3F (A3F) and inhibit HIV-1 replication in THP-1 cells. We show by qRT-PCR that expression levels of A3G and A3F increase in a dose-dependent manner in the presence of a CC5A extract, as does A3G protein levels by Western blot assay. In addition, when the human monocytic cell line THP-1 was treated with CC5A extract, the replication of HIV-1 IIIB was significantly suppressed compared with IIIB replication in untreated THP-1 cells. Knock down of A3G expression in THP-1 cells compromised the ability of CC5A to inhibit HIV-1 IIIB infectivity. Furthermore, SupT1 cells infected with virus produced from CC5A extract-treated THP-1 cells replicated virus with a higher G to A hypermutation rate (a known consequence of A3G activity) than virus used from untreated THP-1 cells. This suggests that S. cristatus CC5A contains a molecule that induces A3G/F expression and thereby inhibits HIV replication. These findings might lead to the discovery of a novel anti-HIV/AIDS therapeutic.
Asunto(s)
Adhesinas Bacterianas/farmacología , Fármacos Anti-VIH/farmacología , Citidina Desaminasa/metabolismo , Citosina Desaminasa/metabolismo , Endopeptidasas/farmacología , VIH-1/fisiología , Replicación Viral/efectos de los fármacos , Desaminasa APOBEC-3G , Adhesinas Bacterianas/química , Fármacos Anti-VIH/química , Línea Celular , Citidina Desaminasa/genética , Endopeptidasas/química , Estabilidad de Enzimas , Regulación de la Expresión Génica/efectos de los fármacos , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Calor , Humanos , Streptococcus/clasificación , Streptococcus/enzimologíaRESUMEN
GB virus type C (GBV-C) is a single-stranded positive-sense RNA virus classified in the Flaviviridae family. Persistent coinfection with GBV-C is associated with lower human immunodeficiency virus type 1 (HIV-1) load, higher CD4(+) T-cell count, and prolonged survival in HIV-1 coinfected patients. The GBV-C envelope glycoprotein E2 has been reported to interfere with HIV-1 entry. In this study, we showed that the expression of GBV-C E2 inhibited HIV-1 Gag assembly and release. Expression of glycosylated GBV-C E2 inhibited HIV-1 Gag precursor processing, resulting in lower production of CAp24 and MAp17, while the overall expression level of the Gag precursor Pr55 remained unchanged. Membrane floatation gradient and indirect immunofluorescence confocal microscopy analysis showed that glycosylated E2 disrupted HIV-1 Gag trafficking to the plasma membrane, resulting in Gag accumulation in subcellular compartments. This interference in HIV-1 Gag trafficking led to diminished HIV-1 particle production, which is a critical step for HIV-1 to infect new host cells. These findings shed light on a novel mechanism used by GBV-C E2 to inhibit HIV-1 replication and may provide insight into new approaches for suppressing HIV-1 replication.
Asunto(s)
Membrana Celular/metabolismo , Virus GB-C/metabolismo , VIH-1/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Recuento de Linfocito CD4 , Membrana Celular/virología , Coinfección/virología , Virus GB-C/genética , Glicosilación , Células HEK293 , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/genética , VIH-1/patogenicidad , Células HeLa , Humanos , Plásmidos/genética , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Transfección , Proteínas del Envoltorio Viral/genética , Carga Viral , Liberación del Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
APOBEC3G, a potent HIV-1 host restriction factor, is overcome by HIV-1 viral infectivity factor (Vif), which induces its polyubiquitination and proteasomal degradation. Here we show that lysine-deficient APOBEC3G with an N-terminal hemagglutinin (HA) tag fusion (HA-A3G20K/R) was resistant to HIV-1 Vif-induced proteasomal degradation. HA-A3G20K/R molecules were packaged into wild-type HIV-1 particles, and HA-A3G20K/R drastically decreased wild-type HIV-1 reverse transcription products and infectivity. We also showed that the N terminus of A3G was a target of polyubiquitination induced by HIV-1 Vif. Thus, fusion of the HA tag to the N terminus of A3G20K/R reduced its polyubiquitination, the likely mechanism for the resistance of this protein to HIV-1 Vif-induced proteasomal degradation. Finding such ways to induce resistance of A3G to Vif may provide new approaches to anti-HIV/AIDS therapy.
Asunto(s)
Citidina Desaminasa/metabolismo , Hemaglutininas/metabolismo , Lisina/genética , Poliubiquitina/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Desaminasa APOBEC-3G , Línea Celular , Citidina Desaminasa/genética , Hemaglutininas/genética , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Proteasomal degradation of APOBEC3G is a critical step for human immunodeficiency virus type 1 (HIV-1) replication. However, the necessity for polyubiquitination of APOBEC3G in this process is still controversial. In this study, we showed that although macaque simian immunodeficiency virus (SIVmac) Vif is more stable than HIV-1 Vif in human cells, SIVmac Vif induces degradation of APBOEC3G as efficiently as HIV-1 Vif. Overexpression of APOBEC3G or lysine-free APOBEC3G stabilized HIV-1 Vif, indicating that APOBEC3G degradation is independent of the degradation of Vif. Furthermore, an in vivo polyubiquitination assay showed that lysine-free APOBEC3G was also polyubiquitinated. These data suggest that polyubiquitination of APOBEC3G, not that of HIV-1 Vif, is crucial for APOBEC3G degradation.
Asunto(s)
Citidina Desaminasa/metabolismo , VIH-1/patogenicidad , Ubiquitinación , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Desaminasa APOBEC-3G , Línea Celular , VIH-1/inmunología , Humanos , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/patogenicidadRESUMEN
BACKGROUND: Recombination between strains of HIV-1 only occurs in individuals with multiple infections, and the incidence of recombinant forms implies that multiple infection is common. Most direct studies indicate that multiple infection is rare. We determined the rate of multiple infection in a longitudinal study of 58 HIV-1 positive participants from The Women's Interagency HIV Study with a richer sampling design than previous direct studies, and we investigated the role of recombination and sampling design on estimating the multiple infection rate. RESULTS: 40% of our sample had multiple HIV-1 infections. This rate of multiple infection is statistically consistent with previous studies once differences in sampling design are taken into account. Injection drug use significantly increased the incidence of multiple infections. In general there was rapid elimination of secondary strains to undetectable levels, but in 3 cases a superinfecting strain displaced the initial infecting strain and in two cases the strains coexisted throughout the study. All but one secondary strain was detected as an inter- and/or intra-genic recombinant. Injection drug use significantly increased the rate of observed recombinants. CONCLUSION: Our multiple infection rate is consistent with rates estimated from the frequency of recombinant forms of HIV-1. The fact that our results are also consistent with previous direct studies that had reported a much lower rate illustrates the critical role of sampling design in estimating this rate. Multiple infection and recombination significantly add to the genetic diversity of HIV-1 and its evolutionary potential, and injection drug use significantly increases both.
Asunto(s)
Infecciones por VIH/virología , VIH-1/genética , Recombinación Genética , Sobreinfección/epidemiología , Adulto , Estudios de Cohortes , ADN Viral/análisis , ADN Viral/genética , Consumidores de Drogas , Femenino , Variación Genética , Infecciones por VIH/complicaciones , Infecciones por VIH/epidemiología , Humanos , Incidencia , Estudios Longitudinales , Virus Reordenados , Factores de Riesgo , Sobreinfección/etiología , Sobreinfección/virología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/análisis , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/análisis , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
OBJECTIVES: To determine if a history of injection drug use influences genotypic protease inhibitor (PI) resistance to antiretroviral agents. METHODS: We assessed the presence of resistance mutations in PI-naive injection drug users (IDUs) and non-IDUs participating in the Women's Interagency HIV Study. Eighteen HIV-infected participants who reported injection drug use before study enrollment and 32 HIV-infected non-IDUs contributed a total of 34 and 65 person-visits, respectively, to analyses. RESULTS: Based on data from multiple clones obtained from different time points from each individual, we determined that primary PI resistance mutations were more frequent among person visits contributed by IDUs (24%) than non-IDUs (8%, P = 0.05). Although neither reached statistical significance, diversity was higher within the protease region among study visits carrying PI-resistant clones at both the nucleotide level (2.66 vs. 2.35; P = 0.08) and at the amino acid level (1.60 vs. 1.32; P = 0.23). Most of the primary resistance mutations could not be detected using the standard population sequencing employed in the clinical setting. Five of 6 individuals in whom clones encoding PI resistance mutations were identified failed PI-containing highly active antiretroviral therapy within 12 months of therapy initiation. CONCLUSIONS: Our findings indicate that more aggressive sampling for resistance mutations among viral clones before highly active antiretroviral therapy initiation might permit selection of more effective treatment, particularly in IDUs.