RESUMEN
It has recently been shown that an early SARS-CoV-2 isolate (NL-02-2020) hijacks interferon-induced transmembrane proteins (IFITMs) for efficient replication in human lung cells, cardiomyocytes, and gut organoids. To date, several "variants of concern" (VOCs) showing increased infectivity and resistance to neutralization have emerged and globally replaced the early viral strains. Here, we determined whether the five current SARS-CoV-2 VOCs (Alpha, Beta, Gamma, Delta, and Omicron) maintained the dependency on IFITM proteins for efficient replication. We found that depletion of IFITM2 strongly reduces viral RNA production by all VOCs in the human epithelial lung cancer cell line Calu-3. Silencing of IFITM1 had modest effects, while knockdown of IFITM3 resulted in an intermediate phenotype. Strikingly, depletion of IFITM2 generally reduced infectious virus production by more than 4 orders of magnitude. In addition, an antibody directed against the N terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in induced pluripotent stem cell (iPSC)-derived alveolar epithelial type II cells, thought to represent major viral target cells in the lung. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are critical cofactors for efficient replication of genuine SARS-CoV-2 VOCs, including the currently dominant Omicron variant. IMPORTANCE Recent data indicate that SARS-CoV-2 requires endogenously expressed IFITM proteins for efficient infection. However, the results were obtained with an early SARS-CoV-2 isolate. Thus, it remained to be determined whether IFITMs are also important cofactors for infection of emerging SARS-CoV-2 VOCs that outcompeted the original strains in the meantime. This includes the Omicron VOC, which currently dominates the pandemic. Here, we show that depletion of endogenous IFITM2 expression almost entirely prevents productive infection of Alpha, Beta, Gamma, Delta, and Omicron SARS-CoV-2 VOCs in human lung cells. In addition, an antibody targeting the N terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells. Our results show that SARS-CoV-2 VOCs, including the currently dominant Omicron variant, are strongly dependent on IFITM2 for efficient replication, suggesting a key proviral role of IFITMs in viral transmission and pathogenicity.
Asunto(s)
Pulmón , Proteínas de la Membrana , SARS-CoV-2 , Replicación Viral , COVID-19/virología , Línea Celular Tumoral , Humanos , Pulmón/virología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Internalización del VirusRESUMEN
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
Innate antiviral factors are essential for effective defense against viral pathogens. However, the identity of major restriction mechanisms remains elusive. Current approaches to discover antiviral factors usually focus on the initial steps of viral replication and are limited to a single round of infection. Here, we engineered libraries of >1500 replication-competent HIV-1 constructs each expressing a single gRNAs to target >500 cellular genes for virus-driven discovery of antiviral factors. Passaging in CD4+ T cells robustly enriched HIV-1 encoding sgRNAs against GRN, CIITA, EHMT2, CEACAM3, CC2D1B and RHOA by >50-fold. Using an HIV-1 library lacking the accessory nef gene, we identified IFI16 as a Nef target. Functional analyses in cell lines and primary CD4+ T cells support that the HIV-driven CRISPR screen identified restriction factors targeting virus entry, transcription, release and infectivity. Our HIV-guided CRISPR technique enables sensitive discovery of physiologically relevant cellular defense factors throughout the entire viral replication cycle.
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Linfocitos T CD4-Positivos , VIH-1 , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Humanos , VIH-1/genética , VIH-1/fisiología , Replicación Viral/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Linfocitos T CD4-Positivos/virología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Células HEK293 , Sistemas CRISPR-Cas , Infecciones por VIH/virología , Infecciones por VIH/genética , Infecciones por VIH/inmunología , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Internalización del VirusRESUMEN
Host restriction factors play key roles in innate antiviral defense, but it remains poorly understood which of them restricts HIV-1 in vivo. Here, we used single-cell transcriptomic analysis to identify host factors associated with HIV-1 control during acute infection by correlating host gene expression with viral RNA abundance within individual cells. Wide sequencing of cells from one participant with the highest plasma viral load revealed that intracellular viral RNA transcription correlates inversely with expression of the gene PTMA, which encodes prothymosin α. This association was genome-wide significant (Padjusted < 0.05) and was validated in 28 additional participants from Thailand and the Americas with HIV-1 CRF01_AE and subtype B infections, respectively. Overexpression of prothymosin α in vitro confirmed that this cellular factor inhibits HIV-1 transcription and infectious virus production. Our results identify prothymosin α as a host factor that restricts HIV-1 infection in vivo, which has implications for viral transmission and cure strategies.
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Infecciones por VIH , VIH-1 , Humanos , VIH-1/genética , Transcriptoma/genética , Infecciones por VIH/genética , ARN ViralRESUMEN
The accessory viral protein R (Vpr) is encoded by all primate lentiviruses. Vpr counteracts DNA repair pathways, modulates viral immune sensing, and induces cell-cycle arrest in cell culture. However, its impact in vivo is controversial. Here, we show that deletion of vpr is associated with delayed viral replication kinetics, rapid innate immune activation, development and maintenance of strong B and T cell responses, and increased neutralizing activity against SIVmac239 in rhesus macaques. All wild-type SIVmac239-infected animals maintained high viral loads, and five of six developed fatal immunodeficiency during â¼80 weeks of follow-up. Lack of Vpr was associated with better preservation of CD4+ T cells, lower viral loads, and an attenuated clinical course of infection in most animals. Our results show that Vpr contributes to efficient viral immune evasion and the full pathogenic potential of SIVmacin vivo. Inhibition of Vpr may improve humoral immune control of viral replication.
RESUMEN
Humans evolved numerous cell-intrinsic restriction factors as a first line of defense against viral pathogens. Typically, they inhibit efficient viral replication and thus prevent viral zoonoses and pandemics. However, viruses show enormous adaptability and are well known for their ability to counteract antiviral mechanisms. Accumulating evidence shows that some viruses are even capable of exploiting antiviral factors for efficient infection. In addition, antiviral factors may exert enhancing effects under specific circumstances. While much progress has been made in understanding the antiviral mechanisms of restriction factors, their proviral effects are poorly defined. Here, we summarize current knowledge on how viral pathogens may exploit otherwise antiviral cellular factors for efficient infection and replication.
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Inmunidad Innata , Virus , Animales , Antivirales/farmacología , Humanos , Replicación Viral , Virus/genéticaRESUMEN
Cm-p5 is a snail-derived antimicrobial peptide, which demonstrated antifungal activity against the pathogenic strains of Candida albicans. Previously we synthetized a cyclic monomer as well as a parallel and an antiparallel dimer of Cm-p5 with improved antifungal activity. Considering the alarming increase of microbial resistance to conventional antibiotics, here we evaluated the antimicrobial activity of these derivatives against multiresistant and problematic bacteria and against important viral agents. The three peptides showed a moderate activity against Pseudomonas aeruginosa, Klebsiella pneumoniae Extended Spectrum ß-Lactamase (ESBL), and Streptococcus agalactiae, with MIC values > 100 µg/mL. They exerted a considerable activity with MIC values between 25-50 µg/mL against Acinetobacter baumanii and Enterococcus faecium. In addition, the two dimers showed a moderate activity against Pseudomonas aeruginosa PA14. The three Cm-p5 derivatives inhibited a virulent extracellular strain of Mycobacterium tuberculosis, in a dose-dependent manner. Moreover, they inhibited Herpes Simplex Virus 2 (HSV-2) infection in a concentration-dependent manner, but had no effect on infection by the Zika Virus (ZIKV) or pseudoparticles of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2). At concentrations of >100 µg/mL, the three new Cm-p5 derivatives showed toxicity on different eukaryotic cells tested. Considering a certain cell toxicity but a potential interesting activity against the multiresistant strains of bacteria and HSV-2, our compounds require future structural optimization.
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Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Antivirales/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Herpesvirus Humano 2/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Antivirales/química , Candida albicans/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Dimerización , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , SARS-CoV-2/efectos de los fármacosRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades most innate immune responses but may still be vulnerable to some. Here, we systematically analyze the impact of SARS-CoV-2 proteins on interferon (IFN) responses and autophagy. We show that SARS-CoV-2 proteins synergize to counteract anti-viral immune responses. For example, Nsp14 targets the type I IFN receptor for lysosomal degradation, ORF3a prevents fusion of autophagosomes and lysosomes, and ORF7a interferes with autophagosome acidification. Most activities are evolutionarily conserved. However, SARS-CoV-2 Nsp15 antagonizes IFN signaling less efficiently than the orthologs of closely related RaTG13-CoV and SARS-CoV-1. Overall, SARS-CoV-2 proteins counteract autophagy and type I IFN more efficiently than type II or III IFN signaling, and infection experiments confirm potent inhibition by IFN-γ and -λ1. Our results define the repertoire and selected mechanisms of SARS-CoV-2 innate immune antagonists but also reveal vulnerability to type II and III IFN that may help to develop safe and effective anti-viral approaches.
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COVID-19/virología , SARS-CoV-2/inmunología , Proteínas Virales/inmunología , Animales , Antivirales/farmacología , Autofagosomas/inmunología , Autofagia/inmunología , COVID-19/inmunología , Línea Celular , Chlorocebus aethiops , Exorribonucleasas/inmunología , Células HEK293 , Células HeLa , Humanos , Evasión Inmune , Inmunidad Innata , Interferón Tipo I/metabolismo , Interferones/metabolismo , Receptor de Interferón alfa y beta/antagonistas & inhibidores , Receptor de Interferón alfa y beta/inmunología , SARS-CoV-2/patogenicidad , Células Vero , Proteínas no Estructurales Virales/inmunologíaRESUMEN
The bat sarbecovirus RaTG13 is a close relative of SARS-CoV-2, the cause of the COVID-19 pandemic. However, this bat virus was most likely unable to directly infect humans since its Spike (S) protein does not interact efficiently with the human ACE2 receptor. Here, we show that a single T403R mutation increases binding of RaTG13 S to human ACE2 and allows VSV pseudoparticle infection of human lung cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S reduces pseudoparticle infection and viral replication. The T403R RaTG13 S is neutralized by sera from individuals vaccinated against COVID-19 indicating that vaccination might protect against future zoonoses. Our data suggest that a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2 by S proteins of bat coronaviruses. This finding could help to better predict the zoonotic potential of animal coronaviruses.
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Enzima Convertidora de Angiotensina 2/química , Unión Proteica , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Animales , COVID-19/virología , Vacunas contra la COVID-19 , Células CACO-2 , Clonación Molecular , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Mutación , Replicón , Especificidad de la Especie , Células Madre , ZoonosisRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) still presents with a dismal prognosis despite intense research. Better understanding of cellular homeostasis could identify druggable targets to improve therapy. Here we propose RAD50-interacting protein 1 (RINT1) as an essential mediator of cellular homeostasis in PDAC. In a cohort of resected PDAC, low RINT1 protein expression correlated significantly with better survival. Accordingly, RINT1 depletion caused severe growth defects in vitro associated with accumulation of DNA double-strand breaks (DSB), G2 cell cycle arrest, disruption of Golgi-endoplasmic reticulum homeostasis, and cell death. Time-resolved transcriptomics corroborated by quantitative proteome and interactome analyses pointed toward defective SUMOylation after RINT1 loss, impairing nucleocytoplasmic transport and DSB response. Subcutaneous xenografts confirmed tumor response by RINT1 depletion, also resulting in a survival benefit when transferred to an orthotopic model. Primary human PDAC organoids licensed RINT1 relevance for cell viability. Taken together, our data indicate that RINT1 loss affects PDAC cell fate by disturbing SUMOylation pathways. Therefore, a RINT1 interference strategy may represent a new putative therapeutic approach. SIGNIFICANCE: These findings provide new insights into the aggressive behavior of PDAC, showing that RINT1 directly correlates with survival in patients with PDAC by disturbing the SUMOylation process, a crucial modification in carcinogenesis.
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Carcinoma Ductal Pancreático , Proteínas de Ciclo Celular/fisiología , Reparación del ADN/genética , Neoplasias Pancreáticas , Sumoilación , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Estudios de Cohortes , Daño del ADN/genética , Femenino , Homeostasis/genética , Humanos , Ratones , Ratones Desnudos , Ratones Transgénicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Procesamiento Proteico-Postraduccional/genética , Sumoilación/genéticaRESUMEN
SARS-CoV-2 is a respiratory pathogen and primarily infects the airway epithelium. As our knowledge about innate immune factors of the respiratory tract against SARS-CoV-2 is limited, we generated and screened a peptide/protein library derived from bronchoalveolar lavage for inhibitors of SARS-CoV-2 spike-driven entry. Analysis of antiviral fractions revealed the presence of α1-antitrypsin (α1AT), a highly abundant circulating serine protease inhibitor. Here, we report that α1AT inhibits SARS-CoV-2 entry at physiological concentrations and suppresses viral replication in cell lines and primary cells including human airway epithelial cultures. We further demonstrate that α1AT binds and inactivates the serine protease TMPRSS2, which enzymatically primes the SARS-CoV-2 spike protein for membrane fusion. Thus, the acute phase protein α1AT is an inhibitor of TMPRSS2 and SARS-CoV-2 entry, and may play an important role in the innate immune defense against the novel coronavirus. Our findings suggest that repurposing of α1AT-containing drugs has prospects for the therapy of COVID-19.
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Tratamiento Farmacológico de COVID-19 , SARS-CoV-2/efectos de los fármacos , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/farmacología , alfa 1-Antitripsina/farmacología , Anticuerpos Antivirales/sangre , Antivirales/farmacología , COVID-19/sangre , Células CACO-2 , Humanos , Inmunoglobulina G/sangre , Simulación del Acoplamiento Molecular , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) can restrict viral pathogens, but pro- and anti-viral activities have been reported for coronaviruses. Here, we show that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. However, endogenous IFITM expression supports efficient infection of SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral infection. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. IFITM-derived peptides and targeting antibodies inhibit SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and are potential targets for therapeutic approaches.