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1.
Nature ; 615(7950): 134-142, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36470304

RESUMEN

Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)1, could represent a new chemoprophylactic approach for COVID-19 that complements vaccination2,3. However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.


Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Receptores Virales , Ácido Ursodesoxicólico , Animales , Humanos , Ratones , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/metabolismo , COVID-19/prevención & control , Receptores Virales/genética , Receptores Virales/metabolismo , Estudios Retrospectivos , SARS-CoV-2/metabolismo , Tratamiento Farmacológico de COVID-19 , Cricetinae , Transcripción Genética , Ácido Ursodesoxicólico/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Organoides/efectos de los fármacos , Organoides/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/metabolismo , Sistema de Registros , Reproducibilidad de los Resultados , Trasplante de Hígado
2.
Nature ; 603(7902): 706-714, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35104837

RESUMEN

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.


Asunto(s)
COVID-19/patología , COVID-19/virología , Fusión de Membrana , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Serina Endopeptidasas/metabolismo , Internalización del Virus , Adulto , Anciano , Anciano de 80 o más Años , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Línea Celular , Membrana Celular/metabolismo , Membrana Celular/virología , Chlorocebus aethiops , Convalecencia , Femenino , Humanos , Sueros Inmunes/inmunología , Intestinos/patología , Intestinos/virología , Pulmón/patología , Pulmón/virología , Masculino , Persona de Mediana Edad , Mutación , Mucosa Nasal/patología , Mucosa Nasal/virología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Técnicas de Cultivo de Tejidos , Virulencia , Replicación Viral
3.
Nature ; 592(7853): 277-282, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33545711

RESUMEN

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.


Asunto(s)
Tratamiento Farmacológico de COVID-19 , COVID-19/terapia , COVID-19/virología , Evolución Molecular , Mutagénesis/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Adenosina Monofosfato/uso terapéutico , Anciano , Alanina/análogos & derivados , Alanina/farmacología , Alanina/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Enfermedad Crónica , Genoma Viral/efectos de los fármacos , Genoma Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Evasión Inmune/efectos de los fármacos , Evasión Inmune/genética , Evasión Inmune/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Tolerancia Inmunológica/inmunología , Inmunización Pasiva , Terapia de Inmunosupresión , Masculino , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Mutación , Filogenia , SARS-CoV-2/inmunología , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Factores de Tiempo , Carga Viral/efectos de los fármacos , Esparcimiento de Virus , Sueroterapia para COVID-19
4.
Nature ; 599(7883): 114-119, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34488225

RESUMEN

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.


Asunto(s)
Evasión Inmune , SARS-CoV-2/crecimiento & desarrollo , SARS-CoV-2/inmunología , Replicación Viral/inmunología , Anticuerpos Neutralizantes/inmunología , Vacunas contra la COVID-19/inmunología , Fusión Celular , Línea Celular , Femenino , Personal de Salud , Humanos , India , Cinética , Masculino , Glicoproteína de la Espiga del Coronavirus/metabolismo , Vacunación
5.
EMBO J ; 41(22): e111653, 2022 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-36161661

RESUMEN

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a great threat to human health. The interplay between the virus and host plays a crucial role in successful virus replication and transmission. Understanding host-virus interactions are essential for the development of new COVID-19 treatment strategies. Here, we show that SARS-CoV-2 infection triggers redistribution of cyclin D1 and cyclin D3 from the nucleus to the cytoplasm, followed by proteasomal degradation. No changes to other cyclins or cyclin-dependent kinases were observed. Further, cyclin D depletion was independent of SARS-CoV-2-mediated cell cycle arrest in the early S phase or S/G2/M phase. Cyclin D3 knockdown by small-interfering RNA specifically enhanced progeny virus titres in supernatants. Finally, cyclin D3 co-immunoprecipitated with SARS-CoV-2 envelope (E) and membrane (M) proteins. We propose that cyclin D3 impairs the efficient incorporation of envelope protein into virions during assembly and is depleted during SARS-CoV-2 infection to restore efficient assembly and release of newly produced virions.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Ciclina D3 , Pandemias , Línea Celular , Virión , Tratamiento Farmacológico de COVID-19
7.
EMBO J ; 37(1): 50-62, 2018 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-29084722

RESUMEN

We report that DNA damage induced by topoisomerase inhibitors, including etoposide (ETO), results in a potent block to HIV-1 infection in human monocyte-derived macrophages (MDM). SAMHD1 suppresses viral reverse transcription (RT) through depletion of cellular dNTPs but is naturally switched off by phosphorylation in a subpopulation of MDM found in a G1-like state. We report that SAMHD1 was activated by dephosphorylation following ETO treatment, along with loss of expression of MCM2 and CDK1, and reduction in dNTP levels. Suppression of infection occurred after completion of viral DNA synthesis, at the step of 2LTR circle and provirus formation. The ETO-induced block was completely rescued by depletion of SAMHD1 in MDM Concordantly, infection by HIV-2 and SIVsm encoding the SAMHD1 antagonist Vpx was insensitive to ETO treatment. The mechanism of DNA damage-induced blockade of HIV-1 infection involved activation of p53, p21, decrease in CDK1 expression, and SAMHD1 dephosphorylation. Therefore, topoisomerase inhibitors regulate SAMHD1 and HIV permissivity at a post-RT step, revealing a mechanism by which the HIV-1 reservoir may be limited by chemotherapeutic drugs.


Asunto(s)
Daño del ADN/efectos de los fármacos , Etopósido/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Macrófagos/efectos de los fármacos , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Replicación Viral/efectos de los fármacos , Células Cultivadas , Infecciones por VIH/virología , Humanos , Macrófagos/metabolismo , Macrófagos/virología , Nucleótidos/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacología
8.
EMBO J ; 36(5): 604-616, 2017 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-28122869

RESUMEN

An unresolved question is how HIV-1 achieves efficient replication in terminally differentiated macrophages despite the restriction factor SAMHD1. We reveal inducible changes in expression of cell cycle-associated proteins including MCM2 and cyclins A, E, D1/D3 in macrophages, without evidence for DNA synthesis or mitosis. These changes are induced by activation of the Raf/MEK/ERK kinase cascade, culminating in upregulation of CDK1 with subsequent SAMHD1 T592 phosphorylation and deactivation of its antiviral activity. HIV infection is limited to these G1-like phase macrophages at the single-cell level. Depletion of SAMHD1 in macrophages decouples the association between infection and expression of cell cycle-associated proteins, with terminally differentiated macrophages becoming highly susceptible to HIV-1. We observe both embryo-derived and monocyte-derived tissue-resident macrophages in a G1-like phase at frequencies approaching 20%, suggesting how macrophages sustain HIV-1 replication in vivo Finally, we reveal a SAMHD1-dependent antiretroviral activity of histone deacetylase inhibitors acting via p53 activation. These data provide a basis for host-directed therapeutic approaches aimed at limiting HIV-1 burden in macrophages that may contribute to curative interventions.


Asunto(s)
Fase G1 , VIH-1/fisiología , Evasión Inmune , Macrófagos/inmunología , Macrófagos/virología , Proteínas de Unión al GTP Monoméricas/metabolismo , Procesamiento Proteico-Postraduccional , Células Cultivadas , VIH-1/inmunología , Humanos , Inmunidad Innata , Fosforilación , Proteína 1 que Contiene Dominios SAM y HD
9.
Clin Infect Dis ; 61(1): 120-8, 2015 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-25778749

RESUMEN

BACKGROUND: Emerging data relating to human immunodeficiency virus type 1 (HIV-1) cure suggest that vaccination to stimulate the host immune response, particularly cytotoxic cells, may be critical to clearing of reactivated HIV-1-infected cells. However, evidence for this approach in humans is lacking, and parameters required for a vaccine are unknown because opportunities to study HIV-1 reactivation are rare. METHODS: We present observations from a HIV-1 elite controller, not treated with combination antiretroviral therapy, who experienced viral reactivation following treatment for myeloma with melphalan and autologous stem cell transplantation. Mathematical modeling was performed using a standard viral dynamic model. Enzyme-linked immunospot, intracellular cytokine staining, and tetramer staining were performed on peripheral blood mononuclear cells; in vitro CD8 T-cell-mediated control of virion production by autologous CD4 T cells was quantified; and neutralizing antibody titers were measured. RESULTS: Viral rebound was measured at 28,000 copies/mL on day 13 post-transplant before rapid decay to <50 copies/mL in 2 distinct phases with t1/2 of 0.71 days and 4.1 days. These kinetics were consistent with an expansion of cytotoxic effector cells and killing of productively infected CD4 T cells. Following transplantation, innate immune cells, including natural killer cells, recovered with virus rebound. However, most striking was the expansion of highly functional HIV-1-specific cytotoxic CD8 T cells, at numbers consistent with those applied in modeling, as virus control was regained. CONCLUSIONS: These observations provide evidence that the human immune response is capable of controlling coordinated global HIV-1 reactivation, remarkably with potency equivalent to combination antiretroviral therapy. These data will inform design of vaccines for use in HIV-1 curative interventions.


Asunto(s)
Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , VIH-1/fisiología , Activación Viral/inmunología , Anticuerpos Neutralizantes/sangre , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/inmunología , Citocinas/análisis , Ensayo de Immunospot Ligado a Enzimas , Anticuerpos Anti-VIH/sangre , Infecciones por VIH/complicaciones , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Melfalán/efectos adversos , Melfalán/uso terapéutico , Persona de Mediana Edad , Modelos Teóricos , Mieloma Múltiple/tratamiento farmacológico , Agonistas Mieloablativos/efectos adversos , Agonistas Mieloablativos/uso terapéutico , Trasplante de Células Madre/efectos adversos , Subgrupos de Linfocitos T/inmunología , Trasplante Autólogo
10.
Traffic ; 13(2): 273-91, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22017400

RESUMEN

In human monocyte-derived macrophages (MDM), human immunodeficiency virus type 1 (HIV-1) assembly takes place primarily on complex intracellular plasma membrane domains connected to the cell surface by closely apposed membrane sheets or narrow channels. Some of the membranes associated with these compartments are decorated by thick (≈30 nm), electron-dense, cytoplasmic coats. Here we show by immunolabelling of ultrathin cryosections that the ß2 integrin CD18, together with the αM and αX integrins (CD11b and CD11c), is clustered at these coated domains, and that the coats themselves contain the cytoskeletal linker proteins talin, vinculin and paxillin that connect the integrin complexes to the actin cytoskeleton. Intracellular plasma membrane-connected compartments (IPMC) with CD18-containing focal adhesion-like coats are also present in uninfected MDM. These compartments become more prominent as the cells mature in tissue culture and their appearance correlates with increased expression of CD18, CD11b/c and paxillin. Depletion of CD18 by RNA interference leads to parallel down-regulation of CD11b and CD11c, as well as of paxillin, and the disappearance of the adhesion-like coats. In addition, CD18 knockdown alters the appearance of virus-containing IPMC in HIV-infected MDM, indicating that the ß2 integrin/focal adhesion-like coat structures are involved in the organization of these compartments.


Asunto(s)
Antígenos CD18/metabolismo , Estructuras de la Membrana Celular/fisiología , Estructuras de la Membrana Celular/virología , VIH-1/crecimiento & desarrollo , Macrófagos/virología , Ensamble de Virus/fisiología , Citoesqueleto de Actina/fisiología , Citoesqueleto de Actina/ultraestructura , Complejo 2 de Proteína Adaptadora/metabolismo , Antígeno CD11b/metabolismo , Antígeno CD11c/metabolismo , Antígenos CD18/genética , Diferenciación Celular/fisiología , Estructuras de la Membrana Celular/ultraestructura , Células Cultivadas , Clatrina/metabolismo , Regulación hacia Abajo/genética , Antígenos VIH/metabolismo , VIH-1/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/ultraestructura , Paxillin/metabolismo , ARN Interferente Pequeño/genética , Talina/metabolismo , Tetraspanina 29/metabolismo , Vinculina/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo
11.
Retrovirology ; 11: 25, 2014 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-24656066

RESUMEN

BACKGROUND: It is now known that clinically derived viruses are most commonly R5 tropic with very low infectivity in macrophages. As these viruses utilize CD4 inefficiently, defective entry has been assumed to be the dominant restriction. The implication is that macrophages are not an important reservoir for the majority of circulating viruses. RESULTS: Macrophage infection by clinical transmitted/founder isolates was 10-100 and 30-450 fold less efficient as compared to YU-2 and BaL respectively. Vpx complementation augmented macrophage infection by non-macrophage tropic viruses to the level of infectivity observed for YU-2 in the absence of Vpx. Augmentation was evident even when Vpx was provided 24 hours post-infection. The entry defect was measured as 2.5-5 fold, with a further 3.5-10 fold block at strong stop and subsequent stages of reverse transcription as compared to YU-2. The overall block to infection was critically dependent on the mechanism of entry as demonstrated by rescue of infection after pseudotyping with VSV-G envelope. Reverse transcription in macrophages could not be enhanced using a panel of cytokines or lipopolysaccharide (LPS). CONCLUSIONS: Although the predominant block to clinical transmitted/founder viruses is post-entry, infectivity is determined by Env-CD4 interactions and can be rescued with VSV-G pseudotyping. This suggests a functional link between the optimal entry pathway taken by macrophage tropic viruses and downstream events required for reverse transcription. Consistent with a predominantly post-entry block, replication of R5 using viruses can be greatly enhanced by Vpx. We conclude therefore that entry is not the limiting step and that macrophages represent clinically relevant reservoirs for 'non-macrophage tropic' viruses.


Asunto(s)
VIH-1/fisiología , Macrófagos/virología , Proteínas Reguladoras y Accesorias Virales/metabolismo , Internalización del Virus , Línea Celular , Humanos , Receptores CCR5/metabolismo , Receptores del VIH/metabolismo , Tropismo Viral
12.
J Virol ; 87(24): 13124-33, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24067975

RESUMEN

Tetherin is a broadly active antiviral effector that works by tethering nascent enveloped virions to a host cell membrane, thus preventing their release. In this study, we demonstrate that herpes simplex virus 1 (HSV-1) is targeted by tetherin. We identify the viral envelope glycoprotein M (gM) as having moderate anti-tetherin activity. We show that gM but not gB or gD efficiently removes tetherin from the plasma membrane and can functionally substitute for the human immunodeficiency virus type 1 (HIV-1) Vpu protein, the prototypic viral tetherin antagonist, in rescuing HIV-1 release from tetherin-expressing cells. Our data emphasize that tetherin is a broadly active antiviral effector and contribute to the emerging hypothesis that viruses must suppress or evade an array of host cell countermeasures in order to establish a productive infection.


Asunto(s)
Antígenos CD/metabolismo , Herpes Simple/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Virales/metabolismo , Antígenos CD/genética , Membrana Celular/metabolismo , Membrana Celular/virología , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Herpes Simple/genética , Herpesvirus Humano 1/genética , Interacciones Huésped-Patógeno , Humanos , Glicoproteínas de Membrana/genética , Unión Proteica , Proteínas Virales/genética
13.
BMC Biol ; 11: 89, 2013 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-23915020

RESUMEN

BACKGROUND: In HIV-1-infected human monocyte-derived macrophages (MDMs), virus particles assemble primarily on intracellularly sequestered plasma membrane domains termed intracellular plasma membrane-connected compartments (IPMCs). Despite their clear role in virus formation, little is known of the organization, composition, dynamics or function of these compartments. RESULTS: We have used amphipathic membrane dyes to reveal the complex three-dimensional structure of IPMCs in whole MDMs and to visualize connections between IPMCs and the cell surface. The observation of similar IPMC structures in both infected and uninfected cells indicates that these compartments are not induced by virus infection, but are present constitutively in MDMs. By expressing a phospholipase Cδ pleckstrin homology domain linked to green fluorescent protein, we demonstrate that IPMCs contain phosphatidylinositol 4,5-bisphosphate. Live cell imaging of cells expressing this probe shows that IPMCs are dynamic, but relatively stable, sub-domains of the plasma membrane. As recent electron microscopy studies indicated that portions of IPMCs are coated with ß2 integrin-containing focal adhesion-like complexes linked to actin, we investigated whether the actin cytoskeleton is required for the organization of IPMCs. In MDMs treated with the actin polymerization inhibitor latrunculin, the normally compact IPMCs dispersed into smaller structures that remained connected to the plasma membrane. Moreover, latrunculin enhanced the release of preformed, mature HIV-1 particles from infected MDMs. CONCLUSIONS: IPMCs are constitutive features of MDMs that are continuous with the plasma membrane and are used as unique sites for the assembly of new virions following infection by HIV-1. A functionally intact actin cytoskeleton is required to maintain the organization of the IPMCs and, in HIV-1-infected cells, perturbation of the actin cytoskeleton influences both the organization of the compartment and the release of sequestered virus.


Asunto(s)
Compartimento Celular , Membrana Celular/virología , VIH-1/fisiología , Membranas Intracelulares/virología , Macrófagos/virología , Ensamble de Virus/fisiología , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compartimento Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colorantes/farmacología , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Fluorescentes Verdes/metabolismo , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Humanos , Procesamiento de Imagen Asistido por Computador , Membranas Intracelulares/efectos de los fármacos , Lípidos/química , Macrófagos/metabolismo , Macrófagos/ultraestructura , Monocitos/citología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Polimerizacion/efectos de los fármacos , Tiazolidinas/farmacología , Ensamble de Virus/efectos de los fármacos
14.
Biochim Biophys Acta Mol Basis Dis ; 1870(5): 167193, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38648902

RESUMEN

SARS-CoV-2 infection can cause severe pneumonia, wherein exacerbated inflammation plays a major role. This is reminiscent of the process commonly termed cytokine storm, a condition dependent on a disproportionated production of cytokines. This state involves the activation of the innate immune response by viral patterns and coincides with the biosynthesis of the biomass required for viral replication, which may overwhelm the capacity of the endoplasmic reticulum and drive the unfolded protein response (UPR). The UPR is a signal transduction pathway composed of three branches that is initiated by a set of sensors: inositol-requiring protein 1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6). These sensors control adaptive processes, including the transcriptional regulation of proinflammatory cytokines. Based on this background, the role of the UPR in SARS-CoV-2 replication and the ensuing inflammatory response was investigated using in vivo and in vitro models of infection. Mice and Syrian hamsters infected with SARS-CoV-2 showed a sole activation of the Ire1α-Xbp1 arm of the UPR associated with a robust production of proinflammatory cytokines. Human lung epithelial cells showed the dependence of viral replication on the expression of UPR-target proteins branching on the IRE1α-XBP1 arm and to a lower extent on the PERK route. Likewise, activation of the IRE1α-XBP1 branch by Spike (S) proteins from different variants of concern was a uniform finding. These results show that the IRE1α-XBP1 system enhances viral replication and cytokine expression and may represent a potential therapeutic target in SARS-CoV-2 severe pneumonia.


Asunto(s)
COVID-19 , Endorribonucleasas , Proteínas Serina-Treonina Quinasas , SARS-CoV-2 , Respuesta de Proteína Desplegada , Replicación Viral , Proteína 1 de Unión a la X-Box , Animales , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , Proteína 1 de Unión a la X-Box/metabolismo , Proteína 1 de Unión a la X-Box/genética , SARS-CoV-2/metabolismo , Humanos , COVID-19/metabolismo , COVID-19/virología , COVID-19/patología , COVID-19/inmunología , Ratones , Mesocricetus , Transducción de Señal , Ratones Endogámicos C57BL , Citocinas/metabolismo , Femenino
15.
Curr Opin Infect Dis ; 26(6): 561-6, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24152764

RESUMEN

PURPOSE OF REVIEW: There has been a shift towards HIV-1 eradication research in the last three years, yet much is still unknown about the precise role that macrophages will play in any such strategy. This review attempts to summarize the latest data on this subject. RECENT FINDINGS: A new generation of histone deacetylase inhibitors, ITF2357, belinostat, givinostat, panobinostat, and the cancer drug JQ1, have been shown to induce viral reactivation in a monocyte cell line. In macrophages chronically infected with HIV-1 in vitro, drugs blocking pre-integration steps have demonstrated poor efficacy in controlling viral replication in comparison to protease inhibitors, thus questioning whether drugs can control this reservoir following histone deacetylase inhibition. Finally, non-human primate data suggest that CD8+ T cells may not be able to clear infected macrophages. SUMMARY: Given these data highlighting the barriers to addressing the macrophage reservoir, functional rather than sterilizing cure may be a realistic goal. More research on macrophages is needed and animal models may prove useful in future HIV-1 eradication studies by offering a clinically relevant way to study macrophage infection in vivo.


Asunto(s)
Antirretrovirales/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Macrófagos/virología , Animales , Reservorios de Enfermedades/virología , VIH-1/fisiología , Humanos , Monocitos/virología , Activación Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacos
16.
Proc Natl Acad Sci U S A ; 106(49): 20889-94, 2009 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-19864625

RESUMEN

Tetherin is an IFN-inducible restriction factor that inhibits HIV-1 particle release in the absence of the HIV-1 countermeasure, viral protein U (Vpu). Although ubiquitous in HIV-1 and simian immunodeficiency viruses from chimpanzees, greater spot nosed monkeys, mustached monkeys, and Mona monkeys, other primate lentiviruses do not encode a Vpu protein. Here we demonstrate that SIV from Tantalus monkeys (SIVtan) encodes an envelope glycoprotein (SIVtan Env) able to counteract tetherin from Tantalus monkeys, rhesus monkeys, sooty mangabeys, and humans, but not from pigs. We show that sensitivity to Vpu but not SIVtan Env can be transferred with the human tetherin transmembrane region. We also identify a mutation in the tetherin extracellular domain, which almost completely abolishes sensitivity of human tetherin to SIVtan Env without compromising antiviral activity or sensitivity to Vpu. SIVtan Env expression results in a reduction of surface tetherin, as well as reduction in tetherin co-localization with mature surface-associated virus. Immuno-electron microscopy reveals co-localization of SIVtan Env with tetherin in intracellular tubulo-vesicular structures, suggesting that tetherin is sequestered away from budding virions at the cell surface. Along with HIV-1 Vpu and SIV Nef, envelope glycoprotein is the third and most broadly active lentiviral-encoded tetherin countermeasure to be described. Our observations emphasize the importance of tetherin in protecting mammals against viral infection and suggest that HIV-1 Vpu inhibitors may select active envelope mutants.


Asunto(s)
Antígenos CD/metabolismo , Espacio Intracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD/química , Antígenos CD/ultraestructura , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Espacio Extracelular/metabolismo , Proteínas Ligadas a GPI , Haplorrinos , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/ultraestructura , Datos de Secuencia Molecular , Mutación Puntual/genética , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas del Envoltorio Viral/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo
17.
Nat Commun ; 13(1): 6131, 2022 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-36253377

RESUMEN

Real-world data on vaccine-elicited neutralising antibody responses for two-dose AZD1222 in African populations are limited. We assessed baseline SARS-CoV-2 seroprevalence and levels of protective neutralizing antibodies prior to vaccination rollout using binding antibodies analysis coupled with pseudotyped virus neutralisation assays in two cohorts from West Africa: Nigerian healthcare workers (n = 140) and a Ghanaian community cohort (n = 527) pre and post vaccination. We found 44 and 28% of pre-vaccination participants showed IgG anti-N positivity, increasing to 59 and 39% respectively with anti-receptor binding domain (RBD) IgG-specific antibodies. Previous IgG anti-N positivity significantly increased post two-dose neutralizing antibody titres in both populations. Serological evidence of breakthrough infection was observed in 8/49 (16%). Neutralising antibodies were observed to wane in both populations, especially in anti-N negative participants with an observed waning rate of 20% highlighting the need for a combination of additional markers to characterise previous infection. We conclude that AZD1222 is immunogenic in two independent West African cohorts with high background seroprevalence and incidence of breakthrough infection in 2021. Waning titres post second dose indicates the need for booster dosing after AZD1222 in the African setting despite hybrid immunity from previous infection.


Asunto(s)
COVID-19 , Vacunas Virales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Formación de Anticuerpos , COVID-19/epidemiología , COVID-19/prevención & control , ChAdOx1 nCoV-19 , Ghana , Humanos , Inmunoglobulina G , SARS-CoV-2 , Estudios Seroepidemiológicos , Vacunación
18.
Cell Rep ; 35(13): 109292, 2021 06 29.
Artículo en Inglés | MEDLINE | ID: mdl-34166617

RESUMEN

We report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike ΔH69/V70 in multiple independent lineages, often occurring after acquisition of receptor binding motif replacements such as N439K and Y453F, known to increase binding affinity to the ACE2 receptor and confer antibody escape. In vitro, we show that, although ΔH69/V70 itself is not an antibody evasion mechanism, it increases infectivity associated with enhanced incorporation of cleaved spike into virions. ΔH69/V70 is able to partially rescue infectivity of spike proteins that have acquired N439K and Y453F escape mutations by increased spike incorporation. In addition, replacement of the H69 and V70 residues in the Alpha variant B.1.1.7 spike (where ΔH69/V70 occurs naturally) impairs spike incorporation and entry efficiency of the B.1.1.7 spike pseudotyped virus. Alpha variant B.1.1.7 spike mediates faster kinetics of cell-cell fusion than wild-type Wuhan-1 D614G, dependent on ΔH69/V70. Therefore, as ΔH69/V70 compensates for immune escape mutations that impair infectivity, continued surveillance for deletions with functional effects is warranted.


Asunto(s)
COVID-19/inmunología , COVID-19/virología , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Línea Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Evasión Inmune , Mutación , Pandemias , Filogenia , Unión Proteica , Recurrencia , SARS-CoV-2/inmunología , Células Vero
19.
Cell Rep ; 30(12): 3972-3980.e5, 2020 03 24.
Artículo en Inglés | MEDLINE | ID: mdl-32209460

RESUMEN

Macrophages exist predominantly in two distinct states, G0 and a G1-like state that is accompanied by phosphorylation of SAMHD1 at T592. Here, we demonstrate that Toll-like receptor 4 (TLR4) activation can potently induce G0 arrest and SAMHD1 antiretroviral activity by an interferon (IFN)-independent pathway. This pathway requires TLR4 engagement with TRIF, but not involvement of TBK1 or IRF3. Exclusive Myd88 activators are unable to trigger G0 arrest or SAMHD1 dephosphorylation, demonstrating this arrest is also Myd88/nuclear factor κB (NF-κB) independent. The G0 arrest is accompanied by p21 upregulation and CDK1 depletion, consistent with the observed SAMHD1 dephosphorylation at T592. Furthermore, we show by SAMHD1 knockdown that the TLR4-activated pathway potently blocks HIV-1 infection in macrophages specifically via SAMHD1. Together, these data demonstrate that macrophages can mobilize an intrinsic cell arrest and anti-viral state by activating TLR4 prior to IFN secretion, thereby highlighting the importance of cell-cycle regulation as a response to pathogen-associated danger signals in macrophages.


Asunto(s)
Antivirales/metabolismo , Puntos de Control del Ciclo Celular , Interferones/metabolismo , Macrófagos/metabolismo , Fase de Descanso del Ciclo Celular , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Transducción de Señal , Receptor Toll-Like 4/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Escherichia coli/metabolismo , Femenino , Infecciones por VIH/patología , Humanos , Lipopolisacáridos/farmacología , Macrófagos/virología , Masculino , Factor 88 de Diferenciación Mieloide/metabolismo , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 5/metabolismo , Regulación hacia Arriba/efectos de los fármacos
20.
Viruses ; 12(8)2020 07 31.
Artículo en Inglés | MEDLINE | ID: mdl-32751972

RESUMEN

Macrophages are the first line of defence against invading pathogens. They play a crucial role in immunity but also in regeneration and homeostasis. Their remarkable plasticity in their phenotypes and function provides them with the ability to quickly respond to environmental changes and infection. Recent work shows that macrophages undergo cell cycle transition from a G0/terminally differentiated state to a G1 state. This G0-to-G1 transition presents a window of opportunity for HIV-1 infection. Macrophages are an important target for HIV-1 but express high levels of the deoxynucleotide-triphosphate hydrolase SAMHD1, which restricts viral DNA synthesis by decreasing levels of dNTPs. While the G0 state is non-permissive to HIV-1 infection, a G1 state is very permissive to HIV-1 infection. This is because macrophages in a G1 state switch off the antiviral restriction factor SAMHD1 by phosphorylation, thereby allowing productive HIV-1 infection. Here, we explore the macrophage cell cycle and the interplay between its regulation and permissivity to HIV-1 infection.


Asunto(s)
Ciclo Celular , VIH-1/fisiología , Macrófagos/fisiología , Macrófagos/virología , Animales , Puntos de Control del Ciclo Celular , Células Cultivadas , Daño del ADN , Fase G1 , Bacterias Gramnegativas/inmunología , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Macrófagos/inmunología , Fosforilación , Fase de Descanso del Ciclo Celular , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo
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