RESUMEN
The unique class of heavy chain-only antibodies, present in Camelidae, can be shrunk to just the variable region of the heavy chain to yield VHHs, also called nanobodies. About one-tenth the size of their full-size counterparts, nanobodies can serve in applications similar to those for conventional antibodies, but they come with a number of signature advantages that find increasing application in biology. They not only function as crystallization chaperones but also can be expressed inside cells as such, or fused to other proteins to perturb the function of their targets, for example, by enforcing their localization or degradation. Their small size also affords advantages when applied in vivo, for example, in imaging applications. Here we review such applications, with particular emphasis on those areas where conventional antibodies would face a more challenging environment.
Asunto(s)
Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Animales , Formación de Anticuerpos , Técnicas de Visualización de Superficie Celular , Ingeniería Genética , Humanos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Anticuerpos de Dominio Único/biosíntesis , Anticuerpos de Dominio Único/uso terapéutico , Relación Estructura-ActividadRESUMEN
Upon detecting pathogens or cell stress, several NOD-like receptors (NLRs) form inflammasome complexes with the adapter ASC and caspase-1, inducing gasdermin D (GSDMD)-dependent cell death and maturation and release of IL-1ß and IL-18. The triggers and activation mechanisms of several inflammasome-forming sensors are not well understood. Here we show that mitochondrial damage activates the NLRP10 inflammasome, leading to ASC speck formation and caspase-1-dependent cytokine release. While the AIM2 inflammasome can also sense mitochondrial demise by detecting mitochondrial DNA (mtDNA) in the cytosol, NLRP10 monitors mitochondrial integrity in an mtDNA-independent manner, suggesting the recognition of distinct molecular entities displayed by the damaged organelles. NLRP10 is highly expressed in differentiated human keratinocytes, in which it can also assemble an inflammasome. Our study shows that this inflammasome surveils mitochondrial integrity. These findings might also lead to a better understanding of mitochondria-linked inflammatory diseases.
Asunto(s)
Citocinas , Inflamasomas , Humanos , Inflamasomas/metabolismo , Caspasa 1/metabolismo , Citocinas/metabolismo , Muerte Celular , ADN Mitocondrial/genética , Interleucina-1beta/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismoRESUMEN
Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.
Asunto(s)
COVID-19/inmunología , Interferón-alfa/inmunología , Células Asesinas Naturales/inmunología , SARS-CoV-2/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Bases , Humanos , Inmunidad Innata/inmunología , Inflamación/inmunología , Interferón-alfa/sangre , Fibrosis Pulmonar/patología , RNA-Seq , Índice de Severidad de la Enfermedad , Transcriptoma/genética , Reino Unido , Estados UnidosRESUMEN
The blood-brain barrier (BBB) protects the central nervous system from infections or harmful substances1; its impairment can lead to or exacerbate various diseases of the central nervous system2-4. However, the mechanisms of BBB disruption during infection and inflammatory conditions5,6 remain poorly defined. Here we find that activation of the pore-forming protein GSDMD by the cytosolic lipopolysaccharide (LPS) sensor caspase-11 (refs. 7-9), but not by TLR4-induced cytokines, mediates BBB breakdown in response to circulating LPS or during LPS-induced sepsis. Mice deficient in the LBP-CD14 LPS transfer and internalization pathway10-12 resist BBB disruption. Single-cell RNA-sequencing analysis reveals that brain endothelial cells (bECs), which express high levels of GSDMD, have a prominent response to circulating LPS. LPS acting on bECs primes Casp11 and Cd14 expression and induces GSDMD-mediated plasma membrane permeabilization and pyroptosis in vitro and in mice. Electron microscopy shows that this features ultrastructural changes in the disrupted BBB, including pyroptotic endothelia, abnormal appearance of tight junctions and vasculature detachment from the basement membrane. Comprehensive mouse genetic analyses, combined with a bEC-targeting adeno-associated virus system, establish that GSDMD activation in bECs underlies BBB disruption by LPS. Delivery of active GSDMD into bECs bypasses LPS stimulation and opens the BBB. In CASP4-humanized mice, Gram-negative Klebsiella pneumoniae infection disrupts the BBB; this is blocked by expression of a GSDMD-neutralizing nanobody in bECs. Our findings outline a mechanism for inflammatory BBB breakdown, and suggest potential therapies for diseases of the central nervous system associated with BBB impairment.
Asunto(s)
Barrera Hematoencefálica , Encéfalo , Células Endoteliales , Gasderminas , Inflamación , Animales , Femenino , Humanos , Masculino , Ratones , Membrana Basal/metabolismo , Membrana Basal/ultraestructura , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/ultraestructura , Barrera Hematoencefálica/virología , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Caspasas Iniciadoras/metabolismo , Dependovirus , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Gasderminas/antagonistas & inhibidores , Gasderminas/metabolismo , Inflamación/patología , Inflamación/metabolismo , Klebsiella pneumoniae/fisiología , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/sangre , Lipopolisacáridos/farmacología , Ratones Endogámicos C57BL , Piroptosis , Sepsis/metabolismo , Sepsis/patología , Sepsis/microbiología , Análisis de la Célula Individual , Uniones Estrechas/metabolismo , Uniones Estrechas/ultraestructuraRESUMEN
Zanoni et al. (2017) show that the LPS receptor CD14 promotes internalization of oxidized phospholipids, a hallmark of dying cells, triggering hyperactivation of dendritic cells and macrophages. Their findings reveal CD14 as a regulator of responses to infection and damage, relaying context-dependent signals that determine inflammatory cell fate decisions.
Asunto(s)
Inflamasomas , Lipopolisacáridos , Amigos , Receptores de Lipopolisacáridos , FagocitosRESUMEN
Pyrin is a cytosolic immune sensor that nucleates an inflammasome in response to inhibition of RhoA by bacterial virulence factors, triggering the release of inflammatory cytokines, including IL-1ß. Gain-of-function mutations in the MEFV gene encoding Pyrin cause autoinflammatory disorders, such as familial Mediterranean fever (FMF) and Pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). To precisely define the role of Pyrin in pathogen detection in human immune cells, we compared initiation and regulation of the Pyrin inflammasome response in monocyte-derived macrophages (hMDM). Unlike human monocytes and murine macrophages, we determined that hMDM failed to activate Pyrin in response to known Pyrin activators Clostridioides difficile (C. difficile) toxins A or B (TcdA or TcdB), as well as the bile acid analogue BAA-473. The Pyrin inflammasome response was enabled in hMDM by prolonged priming with either LPS or type I or II interferons and required an increase in Pyrin expression. Notably, FMF mutations lifted the requirement for prolonged priming for Pyrin activation in hMDM, enabling Pyrin activation in the absence of additional inflammatory signals. Unexpectedly, in the absence of a Pyrin response, we found that TcdB activated the NLRP3 inflammasome in hMDM. These data demonstrate that regulation of Pyrin activation in hMDM diverges from monocytes and highlights its dysregulation in FMF.
Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Fiebre Mediterránea Familiar , Humanos , Ratones , Animales , Pirina/genética , Pirina/metabolismo , Fiebre Mediterránea Familiar/genética , Fiebre Mediterránea Familiar/metabolismo , Inflamasomas/metabolismo , Mutación , Macrófagos/metabolismoRESUMEN
COVID-19 outbreak is the biggest threat to human health in recent history. Currently, there are over 1.5 million related deaths and 75 million people infected around the world (as of 22/12/2020). The identification of virulence factors which determine disease susceptibility and severity in different cell types remains an essential challenge. The serine protease TMPRSS2 has been shown to be important for S protein priming and viral entry, however, little is known about its regulation. SPINT2 is a member of the family of Kunitz type serine protease inhibitors and has been shown to inhibit TMPRSS2. Here, we explored the existence of a co-regulation between SPINT2/TMPRSS2 and found a tightly regulated protease/inhibitor expression balance across tissues. We found that SPINT2 negatively correlates with SARS-CoV-2 expression in Calu-3 and Caco-2 cell lines and was down-regulated in secretory cells from COVID-19 patients. We validated our findings using Calu-3 cell lines and observed a strong increase in viral load after SPINT2 knockdown, while overexpression lead to a drastic reduction of the viral load. Additionally, we evaluated the expression of SPINT2 in datasets from comorbid diseases using bulk and scRNA-seq data. We observed its down-regulation in colon, kidney and liver tumors as well as in alpha pancreatic islets cells from diabetes Type 2 patients, which could have implications for the observed comorbidities in COVID-19 patients suffering from chronic diseases.
Asunto(s)
COVID-19/metabolismo , Glicoproteínas de Membrana/metabolismo , SARS-CoV-2/metabolismo , Internalización del Virus , Células A549 , COVID-19/genética , Células CACO-2 , Humanos , Glicoproteínas de Membrana/genética , SARS-CoV-2/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
PURPOSE: NLRC4-associated autoinflammatory disease (NLRC4-AID) is an autosomal dominant condition presenting with a range of clinical manifestations which can include macrophage activation syndrome (MAS) and severe enterocolitis. We now report the first homozygous mutation in NLRC4 (c.478G > A, p.A160T) causing autoinflammatory disease with immune dysregulation and find that heterozygous carriers in the general population are at increased risk of developing ulcerative colitis. METHODS: Circulating immune cells and inflammatory markers were profiled and historical clinical data interrogated. DNA was extracted and sequenced using standard procedures. Inflammasome activation assays for ASC speck formation, pyroptosis, and IL-1ß/IL-18 secretion confirmed pathogenicity of the mutation in vitro. Genome-wide association of NLRC4 (A160T) with ulcerative colitis was examined using data from the IBD exomes portal. RESULTS: A 60-year-old Brazilian female patient was evaluated for recurrent episodes of systemic inflammation from six months of age. Episodes were characterized by recurrent low-grade fever, chills, oral ulceration, uveitis, arthralgia, and abdominal pain, followed by diarrhea with mucus and variable skin rash. High doses of corticosteroids were somewhat effective in controlling disease and anti-IL-1ß therapy partially controlled symptoms. While on treatment, serum IL-1ß and IL-18 levels remained elevated. Genetic investigations identified a homozygous mutation in NLRC4 (A160T), inherited in a recessive fashion. Increased ASC speck formation and IL-1ß/IL-18 secretion confirmed pathogenicity when NLRC4 (A160T) was analyzed in human cell lines. This allele is significantly enriched in patients with ulcerative colitis: OR 2.546 (95% 1.778-3.644), P = 0.01305. CONCLUSION: NLRC4 (A160T) can either cause recessively inherited autoinflammation and immune dysregulation, or function as a heterozygous risk factor for the development of ulcerative colitis.
Asunto(s)
Colitis Ulcerosa , Enfermedades Autoinflamatorias Hereditarias , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas de Unión al Calcio/genética , Colitis Ulcerosa/diagnóstico , Colitis Ulcerosa/genética , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Inflamasomas/metabolismo , Persona de Mediana EdadRESUMEN
Members of the family of pyrin and HIN domain containing (PYHIN) proteins play an emerging role in innate immunity. While absent in melanoma 2 (AIM2) acts a cytosolic sensor of non-self DNA and plays a key role in inflammasome assembly, the γ-interferon-inducible protein 16 (IFI16) restricts retroviral gene expression by sequestering the transcription factor Sp1. Here, we show that the remaining two human PYHIN proteins, i.e. myeloid cell nuclear differentiation antigen (MNDA) and pyrin and HIN domain family member 1 (PYHIN1 or IFIX) share this antiretroviral function of IFI16. On average, knock-down of each of these three nuclear PYHIN proteins increased infectious HIV-1 yield from human macrophages by more than an order of magnitude. Similarly, knock-down of IFI16 strongly increased virus transcription and production in primary CD4+ T cells. The N-terminal pyrin domain (PYD) plus linker region containing a nuclear localization signal (NLS) were generally required and sufficient for Sp1 sequestration and anti-HIV-1 activity of IFI16, MNDA and PYHIN1. Replacement of the linker region of AIM2 by the NLS-containing linker of IFI16 resulted in a predominantly nuclear localization and conferred direct antiviral activity to AIM2 while attenuating its ability to form inflammasomes. The reverse change caused nuclear-to-cytoplasmic relocalization of IFI16 and impaired its antiretroviral activity but did not result in inflammasome assembly. We further show that the Zn-finger domain of Sp1 is critical for the interaction with IFI16 supporting that pyrin domains compete with DNA for Sp1 binding. Finally, we found that human PYHIN proteins also inhibit Hepatitis B virus and simian vacuolating virus 40 as well as the LINE-1 retrotransposon. Altogether, our data show that IFI16, PYHIN1 and MNDA restrict HIV-1 and other viral pathogens by interfering with Sp1-dependent gene expression and support an important role of nuclear PYHIN proteins in innate antiviral immunity.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Núcleo Celular/metabolismo , Infecciones por VIH/prevención & control , VIH-1/inmunología , Macrófagos/inmunología , Proteínas Nucleares/metabolismo , Factor de Transcripción Sp1/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Núcleo Celular/genética , ADN Viral/genética , Células HEK293 , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/aislamiento & purificación , Células Hep G2 , Humanos , Inmunidad Innata/inmunología , Inflamasomas/genética , Inflamasomas/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Proteínas Nucleares/genética , Factor de Transcripción Sp1/genética , Replicación ViralRESUMEN
OBJECTIVES: Here we investigated a patient with inflammatory corneal intraepithelial dyskeratosis, mucosal inflammation, tooth abnormalities and, eczema to uncover the genetic and immunological basis of the disease. METHODS: On suspicion of an autoinflammatory condition, Sanger sequencing of nucleotide-binding oligomerization domain-like, leucine-rich repeat pyrin domain containing 1 (NLRP1) was performed and combined with an in vitro inflammasome reconstitution assay to measure caspase-1-mediated IL-1ß cleavage, stimulation of patient peripheral blood mononuclear cells (PBMCs) and whole blood to measure IL-1ß, IL-18 production and quantification of apoptosis-associated speck-like protein containing CARD (ASC) speck formation as a measure of inflammasome activation by flow cytometry. RESULTS: Sanger sequencing revealed a novel mutation (c.175G>C, p.A59P; NM_33004.4) in the inflammasome molecule NLRP1 segregating with disease, although with incomplete penetrance, in three generations. We found that patient PBMCs produced increased IL-1ß in response to inflammatory stimuli, as well as increased constitutive levels of IL-18. Moreover, we demonstrate that expression of the identified NLRP1 A59P variant caused spontaneous IL-1ß cleavage to mature IL-1ß. In addition, patient PBMCs responded to NLRP1 stimulation with increased ASC speck formation as a reflection of elevated inflammasome activity. CONCLUSION: We demonstrate that this novel NLRP1 A59P variant caused increased activation of the NLRP1 inflammasome, resulting in constitutively and inducibly elevated IL-1ß and IL-18 synthesis. We suggest the NLRP1 mutation underlies the pathogenesis of this rare autoinflammatory dyskeratotic disease inherited in an autosomal dominant manner with incomplete penetrance in the patient and within the family for several generations.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Enfermedades de la Córnea/genética , Disqueratosis Congénita/genética , Enfermedades Autoinflamatorias Hereditarias/genética , Preescolar , Humanos , Masculino , Mutación , Proteínas NLRRESUMEN
CD47 is an antiphagocytic ligand broadly expressed on normal and malignant tissues that delivers an inhibitory signal through the receptor signal regulatory protein alpha (SIRPα). Inhibitors of the CD47-SIRPα interaction improve antitumor antibody responses by enhancing antibody-dependent cellular phagocytosis (ADCP) in xenograft models. Endogenous expression of CD47 on a variety of cell types, including erythrocytes, creates a formidable antigen sink that may limit the efficacy of CD47-targeting therapies. We generated a nanobody, A4, that blocks the CD47-SIRPα interaction. A4 synergizes with anti-PD-L1, but not anti-CTLA4, therapy in the syngeneic B16F10 melanoma model. Neither increased dosing nor half-life extension by fusion of A4 to IgG2a Fc (A4Fc) overcame the issue of an antigen sink or, in the case of A4Fc, systemic toxicity. Generation of a B16F10 cell line that secretes the A4 nanobody showed that an enhanced response to several immune therapies requires near-complete blockade of CD47 in the tumor microenvironment. Thus, strategies to localize CD47 blockade to tumors may be particularly valuable for immune therapy.
Asunto(s)
Antígeno CD47/antagonistas & inhibidores , Inmunoterapia/métodos , Melanoma Experimental/terapia , Anticuerpos de Dominio Único/uso terapéutico , Anemia/inducido químicamente , Animales , Antígeno CD47/inmunología , Evaluación Preclínica de Medicamentos , Ratones Endogámicos C57BL , Fagocitosis , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Microambiente TumoralRESUMEN
Rapid inflammatory responses to cytosolic threats are mediated by inflammasomes - large macromolecular signalling complexes that control the activation of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18, as well as cell death by pyroptosis. Different inflammasome sensors are activated by diverse direct and indirect signals, and subsequently nucleate the polymerization of the adaptor molecule ASC to form signalling platforms macroscopically observed as ASC specks. Caspase-1 is autocatalytically activated at these sites and subsequently matures pro-inflammatory cytokines and the pore-forming effector molecule gasdermin D. While most molecules and basic assembly principles have been deduced from reductionist experimental systems, we still lack fundamental information on the structure and regulation of these complexes in their physiological environment and in the interplay with other signalling pathways. In this review, novel experimental approaches are proposed, including some that rely on nanobodies and single domain antibodies, to understand inflammasome assembly and regulation in the context of the relevant tissues or cells.
Asunto(s)
Inflamasomas/fisiología , Transducción de Señal/fisiología , Animales , Anticuerpos/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/fisiopatología , Anticuerpos de Dominio Único/metabolismoRESUMEN
The transcription and replication machinery of negative-stranded RNA viruses presents a possible target for interference in the viral life cycle. We demonstrate the validity of this concept through the use of cytosolically expressed single-domain antibody fragments (VHHs) that protect cells from a lytic infection with vesicular stomatitis virus (VSV) by targeting the viral nucleoprotein N. We define the binding sites for two such VHHs, 1004 and 1307, by X-ray crystallography to better understand their inhibitory properties. We found that VHH 1307 competes with the polymerase cofactor P for binding and thus inhibits replication and mRNA transcription, while binding of VHH 1004 likely only affects genome replication. The functional relevance of these epitopes is confirmed by the isolation of escape mutants able to replicate in the presence of the inhibitory VHHs. The escape mutations allow identification of the binding site of a third VHH that presumably competes with P for binding at another site than 1307. Collectively, these binding sites uncover different features on the N protein surface that may be suitable for antiviral intervention.
Asunto(s)
Anticuerpos Antivirales/metabolismo , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/inmunología , Anticuerpos de Dominio Único/metabolismo , Virus de la Estomatitis Vesicular Indiana/fisiología , Replicación Viral , Células A549 , Animales , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Sitios de Unión , Cristalografía por Rayos X , Replicación del ADN , Humanos , Mutación , Proteínas de la Nucleocápside/metabolismo , ARN Viral , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Transcripción Genética , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacos , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/inmunologíaRESUMEN
The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.
Asunto(s)
Candida albicans , Candidiasis/inmunología , Interacciones Huésped-Patógeno/inmunología , Fagocitosis/fisiología , Esfingolípidos/biosíntesis , Animales , Candida albicans/inmunología , Línea Celular , Cromatografía en Capa Delgada , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Modelos Animales de Enfermedad , Citometría de Flujo , Técnicas de Inactivación de Genes , Humanos , Espectrometría de Masas , RatonesRESUMEN
UNLABELLED: Perturbation of protein-protein interactions relies mostly on genetic approaches or on chemical inhibition. Small RNA viruses, such as influenza A virus, do not easily lend themselves to the former approach, while chemical inhibition requires that the target protein be druggable. A lack of tools thus constrains the functional analysis of influenza virus-encoded proteins. We generated a panel of camelid-derived single-domain antibody fragments (VHHs) against influenza virus nucleoprotein (NP), a viral protein essential for nuclear trafficking and packaging of the influenza virus genome. We show that these VHHs can target NP in living cells and perturb NP's function during infection. Cytosolic expression of NP-specific VHHs (αNP-VHHs) disrupts virus replication at an early stage of the life cycle. Based on their specificity, these VHHs fall into two distinct groups. Both prevent nuclear import of the viral ribonucleoprotein (vRNP) complex without disrupting nuclear import of NP alone. Different stages of the virus life cycle thus rely on distinct nuclear localization motifs of NP. Their molecular characterization may afford new means of intervention in the virus life cycle. IMPORTANCE: Many proteins encoded by RNA viruses are refractory to manipulation due to their essential role in replication. Thus, studying their function and determining how to disrupt said function through pharmaceutical intervention are difficult. We present a novel method based on single-domain-antibody technology that permits specific targeting and disruption of an essential influenza virus protein in the absence of genetic manipulation of influenza virus itself. Characterization of such interactions may help identify new targets for pharmaceutical intervention. This approach can be extended to study proteins encoded by other viral pathogens.
Asunto(s)
Núcleo Celular/química , Núcleo Celular/virología , Virus de la Influenza A/fisiología , Proteínas de Unión al ARN/análisis , Anticuerpos de Dominio Único/metabolismo , Proteínas del Núcleo Viral/análisis , Virología/métodos , Replicación Viral , Animales , Línea Celular , Perros , Expresión Génica , Biología Molecular/métodos , Proteínas de la Nucleocápside , Anticuerpos de Dominio Único/genéticaRESUMEN
ER-resident proteins destined for degradation are dislocated into the cytosol by components of the ER quality control machinery for proteasomal degradation. Dislocation substrates are ubiquitylated in the cytosol by E2 ubiquitin-conjugating/E3 ligase complexes. UBE2J1 is one of the well-characterized E2 enzymes that participate in this process. However, the physiological function of Ube2j1 is poorly defined. We find that Ube2j1(-/-) mice have reduced viability and fail to thrive early after birth. Male Ube2j1(-/-) mice are sterile due to a defect in late spermatogenesis. Ultrastructural analysis shows that removal of the cytoplasm is incomplete in Ube2j1(-/-) elongating spermatids, compromising the release of mature elongate spermatids into the lumen of the seminiferous tubule. Our findings identify an essential function for the ubiquitin-proteasome-system in spermiogenesis and define a novel, non-redundant physiological function for the dislocation step of ER quality control.
Asunto(s)
Espermatogénesis , Enzimas Ubiquitina-Conjugadoras/metabolismo , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Línea Celular , Retículo Endoplásmico/metabolismo , Inmunoglobulinas/metabolismo , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Masculino , Ratones , Espermátides/citología , Espermátides/patología , Enzimas Ubiquitina-Conjugadoras/deficiencia , Respuesta de Proteína Desplegada , Regulación hacia ArribaRESUMEN
Multiple omics analyzes of Vaccinia virus (VACV) infection have defined molecular characteristics of poxvirus biology. However, little is known about the monkeypox (mpox) virus (MPXV) in humans, which has a different disease manifestation despite its high sequence similarity to VACV. Here, we perform an in-depth multi-omics analysis of the transcriptome, proteome, and phosphoproteome signatures of MPXV-infected primary human fibroblasts to gain insights into the virus-host interplay. In addition to expected perturbations of immune-related pathways, we uncover regulation of the HIPPO and TGF-ß pathways. We identify dynamic phosphorylation of both host and viral proteins, which suggests that MAPKs are key regulators of differential phosphorylation in MPXV-infected cells. Among the viral proteins, we find dynamic phosphorylation of H5 that influenced the binding of H5 to dsDNA. Our extensive dataset highlights signaling events and hotspots perturbed by MPXV, extending the current knowledge on poxviruses. We use integrated pathway analysis and drug-target prediction approaches to identify potential drug targets that affect virus growth. Functionally, we exemplify the utility of this approach by identifying inhibitors of MTOR, CHUK/IKBKB, and splicing factor kinases with potent antiviral efficacy against MPXV and VACV.
Asunto(s)
Fibroblastos , Monkeypox virus , Mpox , Proteínas Virales , Humanos , Monkeypox virus/genética , Fosforilación , Mpox/virología , Mpox/metabolismo , Fibroblastos/virología , Fibroblastos/metabolismo , Proteínas Virales/metabolismo , Proteínas Virales/genética , Proteoma/metabolismo , Interacciones Huésped-Patógeno , Transducción de Señal , Proteómica/métodos , Transcriptoma , Antivirales/farmacología , MultiómicaRESUMEN
Recent studies have revealed new insights into the endocytosis of vaccinia virus (VACV). However, the mechanism of fusion between viral and cellular membranes remains unknown. We developed a microfluidic device with a cell-trap array for immobilization of individual cells, with which we analyzed the acid-dependent fusion of single virions. VACV particles incorporating enhanced green fluorescent protein (EGFP) and labeled with self-quenching concentrations of R18 membrane dye were used in combination with total internal reflection fluorescence microscopy to measure the kinetics of R18 dequenching and thus single hemifusion events initiated by a fast low-pH trigger. These studies revealed unexpectedly long lag phases between pH change and hemifusion. In addition, we found that EGFP fluorescence in the virus was quenched upon acidification, indicating that protons could access the virus core, possibly through a proton channel. In a fraction of virus particles, EGFP fluorescence was recovered, presumably after fusion-pore formation and exposure of the core to the physiological pH of the host-cell cytosol. Given that virus-encoded cation channels play a crucial role in the life cycle of many viruses and can serve as antiviral drug targets, further investigations into a potential VACV viroporin are justified. Our findings indicate that the microfluidic device described may be highly beneficial to similar studies requiring fast kinetic measurements.