RESUMO
Many COVID-19 patients suffer from gastrointestinal symptoms and impaired intestinal barrier function is thought to play a key role in Long COVID. Despite its importance, the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on intestinal epithelia is poorly understood. To address this, we established an intestinal barrier model integrating epithelial Caco-2 cells, mucus-secreting HT29 cells and Raji cells. This gut epithelial model allows efficient differentiation of Caco-2 cells into microfold-like cells, faithfully mimics intestinal barrier function, and is highly permissive to SARS-CoV-2 infection. Early strains of SARS-CoV-2 and the Delta variant replicated with high efficiency, severely disrupted barrier function, and depleted tight junction proteins, such as claudin-1, occludin, and ZO-1. In comparison, Omicron subvariants also depleted ZO-1 from tight junctions but had fewer damaging effects on mucosal integrity and barrier function. Remdesivir, the fusion inhibitor EK1 and the transmembrane serine protease 2 inhibitor Camostat inhibited SARS-CoV-2 replication and thus epithelial barrier damage, while the Cathepsin inhibitor E64d was ineffective. Our results support that SARS-CoV-2 disrupts intestinal barrier function but further suggest that circulating Omicron variants are less damaging than earlier viral strains.
Assuntos
COVID-19 , Mucosa Intestinal , SARS-CoV-2 , Junções Íntimas , Replicação Viral , Humanos , SARS-CoV-2/patogenicidade , Células CACO-2 , COVID-19/virologia , COVID-19/patologia , Mucosa Intestinal/virologia , Mucosa Intestinal/patologia , Junções Íntimas/virologia , Alanina/análogos & derivados , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Antivirais/farmacologia , Células HT29 , Ocludina/metabolismo , Ocludina/genética , Monofosfato de Adenosina/análogos & derivadosRESUMO
Dysregulation of the CXCL12/CXCR4 axis is implicated in autoimmune, inflammatory, and oncogenic diseases, positioning CXCR4 as a pivotal therapeutic target. We evaluated optimized variants of the specific endogenous CXCR4 antagonist, EPI-X4, addressing existing challenges in stability and potency. Our structure-activity relationship study investigates the conjugation of EPI-X4 derivatives with long-chain fatty acids, enhancing serum albumin interaction and receptor affinity. Molecular dynamic simulations revealed that the lipid moieties stabilize the peptide-receptor interaction through hydrophobic contacts at the receptor's N-terminus, anchoring the lipopeptide within the CXCR4 binding pocket and maintaining essential receptor interactions. Accordingly, lipidation resulted in increased receptor affinities and antagonistic activities. Additionally, by interacting with human serum albumin lipidated EPI-X4 derivatives displayed sustained stability in human plasma and extended circulation times in vivo. Selected candidates showed significant therapeutic potential in human retinoblastoma cells in vitro and in ovo, with our lead derivative exhibiting higher efficacies compared to its non-lipidated counterpart. This study not only elucidates the optimization trajectory for EPI-X4 derivatives but also underscores the intricate interplay between stability and efficacy, crucial for delineating their translational potential in clinical applications.
RESUMO
Viral infections pose a significant threat to human health, and effective antiviral strategies are urgently needed. Antiviral peptides have emerged as a promising class of therapeutic agents due to their unique properties and mechanisms of action. While effective on their own, combining antiviral peptides may allow us to enhance their potency and to prevent viral resistance. Here, we developed an orthogonal chemical strategy to prepare a heterodimeric peptide conjugate assembled on a protein-based nanoplatform. Specifically, we combined the optimized version of two peptides inhibiting HIV-1 by distinct mechanisms. Virus-inhibitory peptide (VIRIP) is a 20 amino acid fragment of α1-antitrypsin that inhibits HIV-1 by targeting the gp41 fusion peptide. Endogenous peptide inhibitor of CXCR4 (EPI-X4) is a 16-residue fragment of human serum albumin that prevents HIV-1 entry by binding to the viral CXCR4 co-receptor. Optimized forms of both peptides are assembled on supramolecular nanoplatforms through the streptavidin-biotin interaction. We show that the construct consisting of the two different peptides (SAv-VIR-102C9-EPI-X4 JM#173-C) shows increased activity against CCR5- and CXCR4-tropic HIV-1 variants. Our results are a proof of concept that peptides with different modes of action can be assembled on nanoplatforms to enhance their antiviral activity.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/prevenção & controle , Peptídeos/farmacologia , Albumina Sérica Humana , AntiviraisRESUMO
Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.
Assuntos
COVID-19 , Infecção por Zika virus , Zika virus , Humanos , Peptídeos , Amiloide/química , Antibacterianos/farmacologia , HemoglobinasRESUMO
SARS-CoV-2 Omicron rapidly outcompeted other variants and currently dominates the COVID-19 pandemic. Its enhanced transmission and immune evasion are thought to be driven by numerous mutations in the Omicron Spike protein. Here, we systematically introduced BA.1 and/or BA.2 Omicron Spike mutations into the ancestral Spike protein and examined the impacts on Spike function, processing, and susceptibility to neutralization. Individual mutations of S371F/L, S375F, and T376A in the ACE2-receptor-binding domain as well as Q954H and N969K in the hinge region 1 impaired infectivity, while changes to G339D, D614G, N764K, and L981F moderately enhanced it. Most mutations in the N-terminal region and receptor-binding domain reduced the sensitivity of the Spike protein to neutralization by sera from individuals vaccinated with the BNT162b2 vaccine and by therapeutic antibodies. Our results represent a systematic functional analysis of Omicron Spike adaptations that have allowed this SARS-CoV-2 variant to dominate the current pandemic.
Assuntos
COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacina BNT162 , Humanos , Pandemias , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas do Envelope ViralRESUMO
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.
Assuntos
Pulmão , Proteínas de Membrana , SARS-CoV-2 , Replicação Viral , COVID-19/virologia , Linhagem Celular Tumoral , Humanos , Pulmão/virologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Internalização do VírusRESUMO
The discovery of the G-protein coupled-receptor (GPCR) CXCR4 as a major coreceptor of HIV-1 entry about three decades ago explained why the chemokine SDF-1/CXCL12 inhibits specific viral strains. The knowledge that RANTES, MlP-1α, and MlP-1ß specifically inhibit other primary HIV-1 strains allowed the rapid discovery of CCR5 as second major viral coreceptor and explained why individuals with deletions in CCR5 are protected against sexual HIV-1 transmission. Here, we provide an update on endogenous ligands of GPCRs that act as endogenous inhibitors of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) entry. In addition, we summarize the development of optimized derivatives of endogenous GPCR ligands and their perspectives as antiviral agents and beyond. Finally, we provide examples for other endogenous peptides that may contribute to our innate immune defense against HIV-1 and other viral pathogens and offer prospects for preventive or therapeutic development.
Assuntos
Inibidores da Fusão de HIV , Infecções por HIV , HIV-1 , Animais , Inibidores da Fusão de HIV/farmacologia , Inibidores da Fusão de HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , HIV-1/fisiologia , HIV-2/metabolismo , HIV-2/fisiologia , Humanos , Ligantes , Peptídeos/uso terapêutico , Receptores CCR5 , Receptores Acoplados a Proteínas G/uso terapêutico , Transdução de Sinais , Vírus da Imunodeficiência SímiaRESUMO
Primary airway epithelial cells (pAECs) cultivated at air-liquid interface (ALI) conditions are widely used as surrogates for human in vivo epithelia. To extend the proliferative capacity and to enable serially passaging of pAECs, conditional reprogramming (cr) has been employed in recent years. However, ALI epithelia derived from cr cells often display functional changes with increasing passages. This highlights the need for thorough validation of the ALI cultures for the respective application. In our study, we evaluated the use of serially passaged cr nasal epithelial cells (crNECs) as a model to study SARS-CoV-2 infection and effects on ion and water transport. NECs were obtained from healthy individuals and cultivated as ALI epithelia derived from passages 1, 2, 3, and 5. We compared epithelial differentiation, ion and water transport, and infection with SARS-CoV-2 between passages. Our results show that epithelia maintained major differentiation characteristics and physiological ion and water transport properties through all passages. However, the frequency of ciliated cells, short circuit currents reflecting epithelial Na+ channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) activity and expression of aquaporin 3 and 5 decreased gradually over passages. crNECs also expressed SARS-CoV-2 receptors angiotensin converting enzyme 2 (ACE2) and transmembrane serin2 protease 2 (TMPRSS2) across all passages and allowed SARS-CoV-2 replication in all passages. In summary, we provide evidence that passaged crNECs provide an appropriate model to study SARS-CoV-2 infection and also epithelial transport function when considering some limitations that we defined herein.
Assuntos
COVID-19 , Diferenciação Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Humanos , Recém-Nascido , SARS-CoV-2RESUMO
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.
Assuntos
Enzima de Conversão de Angiotensina 2/genética , Antígenos de Diferenciação/genética , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes/farmacologia , Antígenos de Diferenciação/metabolismo , Sítios de Ligação , COVID-19/virologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Interferon beta/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral/efeitos dos fármacosRESUMO
Guanylate-binding protein (GBP) 5 is an interferon (IFN)-inducible cellular factor reducing HIV-1 infectivity by an incompletely understood mechanism. Here, we show that this activity is shared by GBP2, but not by other members of the human GBP family. GBP2/5 decrease the activity of the cellular proprotein convertase furin, which mediates conversion of the HIV-1 envelope protein (Env) precursor gp160 into mature gp120 and gp41. Because this process primes HIV-1 Env for membrane fusion, viral particles produced in the presence of GBP2/5 are poorly infectious due to increased incorporation of non-functional gp160. Furin activity is critical for the processing of envelope glycoproteins of many viral pathogens. Consistently, GBP2/5 also inhibit Zika, measles, and influenza A virus replication and decrease infectivity of viral particles carrying glycoproteins of Marburg and murine leukemia viruses. Collectively, our results show that GPB2/5 exert broad antiviral activity by suppressing the activity of the virus-dependency factor furin.