RESUMEN
The human seasonal coronavirus HKU1-CoV, which causes common colds worldwide, relies on the sequential binding to surface glycans and transmembrane serine protease 2 (TMPRSS2) for entry into target cells. TMPRSS2 is synthesized as a zymogen that undergoes autolytic activation to process its substrates. Several respiratory viruses, in particular coronaviruses, use TMPRSS2 for proteolytic priming of their surface spike protein to drive membrane fusion upon receptor binding. We describe the crystal structure of the HKU1-CoV receptor binding domain in complex with TMPRSS2, showing that it recognizes residues lining the catalytic groove. Combined mutagenesis of interface residues and comparison across species highlight positions 417 and 469 as determinants of HKU1-CoV host tropism. The structure of a receptor-blocking nanobody in complex with zymogen or activated TMPRSS2 further provides the structural basis of TMPRSS2 activating conformational change, which alters loops recognized by HKU1-CoV and dramatically increases binding affinity.
Asunto(s)
Serina Endopeptidasas , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/química , Humanos , Cristalografía por Rayos X , Coronavirus/metabolismo , Coronavirus/química , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Modelos Moleculares , Unión Proteica , Células HEK293 , Animales , Activación Enzimática , Internalización del VirusRESUMEN
The entry of coronaviruses is initiated by spike recognition of host cellular receptors, involving proteinaceous and/or glycan receptors. Recently, TMPRSS2 was identified as the proteinaceous receptor for HCoV-HKU1 alongside sialoglycan as a glycan receptor. However, the underlying mechanisms for viral entry remain unknown. Here, we investigated the HCoV-HKU1C spike in the inactive, glycan-activated, and functionally anchored states, revealing that sialoglycan binding induces a conformational change of the NTD and promotes the neighboring RBD of the spike to open for TMPRSS2 recognition, exhibiting a synergistic mechanism for the entry of HCoV-HKU1. The RBD of HCoV-HKU1 features an insertion subdomain that recognizes TMPRSS2 through three previously undiscovered interfaces. Furthermore, structural investigation of HCoV-HKU1A in combination with mutagenesis and binding assays confirms a conserved receptor recognition pattern adopted by HCoV-HKU1. These studies advance our understanding of the complex viral-host interactions during entry, laying the groundwork for developing new therapeutics against coronavirus-associated diseases.
Asunto(s)
Serina Endopeptidasas , Glicoproteína de la Espiga del Coronavirus , Internalización del Virus , Humanos , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Polisacáridos/metabolismo , Polisacáridos/química , Células HEK293 , Unión Proteica , Receptores Virales/metabolismo , Receptores Virales/química , Coronavirus/metabolismo , Modelos MolecularesRESUMEN
BA.2.86, a recently identified descendant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 sublineage, contains â¼35 mutations in the spike (S) protein and spreads in multiple countries. Here, we investigated whether the virus exhibits altered biological traits, focusing on S protein-driven viral entry. Employing pseudotyped particles, we show that BA.2.86, unlike other Omicron sublineages, enters Calu-3 lung cells with high efficiency and in a serine- but not cysteine-protease-dependent manner. Robust lung cell infection was confirmed with authentic BA.2.86, but the virus exhibited low specific infectivity. Further, BA.2.86 was highly resistant against all therapeutic antibodies tested, efficiently evading neutralization by antibodies induced by non-adapted vaccines. In contrast, BA.2.86 and the currently circulating EG.5.1 sublineage were appreciably neutralized by antibodies induced by the XBB.1.5-adapted vaccine. Collectively, BA.2.86 has regained a trait characteristic of early SARS-CoV-2 lineages, robust lung cell entry, and evades neutralizing antibodies. However, BA.2.86 exhibits low specific infectivity, which might limit transmissibility.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , SARS-CoV-2 , Humanos , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , Caspasas/metabolismo , COVID-19/inmunología , COVID-19/virología , Pulmón/virología , SARS-CoV-2/clasificación , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , Internalización del Virus , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
While CD4+ T cell depletion is key to disease progression in people living with HIV and SIV-infected macaques, the mechanisms underlying this depletion remain incompletely understood, with most cell death involving uninfected cells. In contrast, SIV infection of "natural" hosts such as sooty mangabeys does not cause CD4+ depletion and AIDS despite high-level viremia. Here, we report that the CARD8 inflammasome is activated immediately after HIV entry by the viral protease encapsulated in incoming virions. Sensing of HIV protease activity by CARD8 leads to rapid pyroptosis of quiescent cells without productive infection, while T cell activation abolishes CARD8 function and increases permissiveness to infection. In humanized mice reconstituted with CARD8-deficient cells, CD4+ depletion is delayed despite high viremia. Finally, we discovered loss-of-function mutations in CARD8 from "natural hosts," which may explain the peculiarly non-pathogenic nature of these infections. Our study suggests that CARD8 drives CD4+ T cell depletion during pathogenic HIV/SIV infections.
Asunto(s)
Infecciones por VIH , Inflamasomas , Síndrome de Inmunodeficiencia Adquirida del Simio , Animales , Humanos , Ratones , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Progresión de la Enfermedad , Infecciones por VIH/patología , Inflamasomas/metabolismo , Proteínas de Neoplasias/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Virus de la Inmunodeficiencia de los Simios/fisiología , Viremia , VIH/fisiologíaRESUMEN
SARS-CoV-2 is associated with broad tissue tropism, a characteristic often determined by the availability of entry receptors on host cells. Here, we show that TMEM106B, a lysosomal transmembrane protein, can serve as an alternative receptor for SARS-CoV-2 entry into angiotensin-converting enzyme 2 (ACE2)-negative cells. Spike substitution E484D increased TMEM106B binding, thereby enhancing TMEM106B-mediated entry. TMEM106B-specific monoclonal antibodies blocked SARS-CoV-2 infection, demonstrating a role of TMEM106B in viral entry. Using X-ray crystallography, cryogenic electron microscopy (cryo-EM), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we show that the luminal domain (LD) of TMEM106B engages the receptor-binding motif of SARS-CoV-2 spike. Finally, we show that TMEM106B promotes spike-mediated syncytium formation, suggesting a role of TMEM106B in viral fusion. Together, our findings identify an ACE2-independent SARS-CoV-2 infection mechanism that involves cooperative interactions with the receptors heparan sulfate and TMEM106B.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Receptores Virales/metabolismo , Internalización del Virus , Unión Proteica , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Simian arteriviruses are endemic in some African primates and can cause fatal hemorrhagic fevers when they cross into primate hosts of new species. We find that CD163 acts as an intracellular receptor for simian hemorrhagic fever virus (SHFV; a simian arterivirus), a rare mode of virus entry that is shared with other hemorrhagic fever-causing viruses (e.g., Ebola and Lassa viruses). Further, SHFV enters and replicates in human monocytes, indicating full functionality of all of the human cellular proteins required for viral replication. Thus, simian arteriviruses in nature may not require major adaptations to the human host. Given that at least three distinct simian arteriviruses have caused fatal infections in captive macaques after host-switching, and that humans are immunologically naive to this family of viruses, development of serology tests for human surveillance should be a priority.
Asunto(s)
Arterivirus , Fiebres Hemorrágicas Virales , Animales , Arterivirus/fisiología , Fiebres Hemorrágicas Virales/veterinaria , Fiebres Hemorrágicas Virales/virología , Humanos , Macaca , Primates , Zoonosis Virales , Internalización del Virus , Replicación ViralRESUMEN
Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAPD3) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAPD3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.
Asunto(s)
Interacciones Huésped-Patógeno , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Virus de la Fiebre del Valle del Rift/fisiología , Internalización del Virus , Animales , Especificidad de Anticuerpos/inmunología , Secuencia de Bases , Encéfalo/patología , Encéfalo/virología , Sistemas CRISPR-Cas/genética , Membrana Celular/metabolismo , Células Cultivadas , Glicoproteínas/metabolismo , Glicosaminoglicanos/metabolismo , Glicosilación , Humanos , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL/metabolismo , Ligandos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/deficiencia , Glicoproteínas de Membrana/metabolismo , Ratones , Unión Proteica , Desnaturalización Proteica , Fiebre del Valle del Rift/patología , Fiebre del Valle del Rift/prevención & control , Fiebre del Valle del Rift/virología , Virus de la Fiebre del Valle del Rift/inmunologíaRESUMEN
Human cytomegalovirus (HCMV) infects the majority of the human population and represents the leading viral cause of congenital birth defects. HCMV utilizes the glycoproteins gHgLgO (Trimer) to bind to platelet-derived growth factor receptor alpha (PDGFRα) and transforming growth factor beta receptor 3 (TGFßR3) to gain entry into multiple cell types. This complex is targeted by potent neutralizing antibodies and represents an important candidate for therapeutics against HCMV. Here, we determine three cryogenic electron microscopy (cryo-EM) structures of the trimer and the details of its interactions with four binding partners: the receptor proteins PDGFRα and TGFßR3 as well as two broadly neutralizing antibodies. Trimer binding to PDGFRα and TGFßR3 is mutually exclusive, suggesting that they function as independent entry receptors. In addition, Trimer-PDGFRα interaction has an inhibitory effect on PDGFRα signaling. Our results provide a framework for understanding HCMV receptor engagement, neutralization, and the development of anti-viral strategies against HCMV.
Asunto(s)
Citomegalovirus/química , Glicoproteínas de Membrana/química , Proteínas del Envoltorio Viral/química , Internalización del Virus , Microscopía por Crioelectrón , Citomegalovirus/fisiología , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Proteoglicanos/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/química , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas del Envoltorio Viral/metabolismoRESUMEN
The global spread of SARS-CoV-2/COVID-19 is devastating health systems and economies worldwide. Recombinant or vaccine-induced neutralizing antibodies are used to combat the COVID-19 pandemic. However, the recently emerged SARS-CoV-2 variants B.1.1.7 (UK), B.1.351 (South Africa), and P.1 (Brazil) harbor mutations in the viral spike (S) protein that may alter virus-host cell interactions and confer resistance to inhibitors and antibodies. Here, using pseudoparticles, we show that entry of all variants into human cells is susceptible to blockade by the entry inhibitors soluble ACE2, Camostat, EK-1, and EK-1-C4. In contrast, entry of the B.1.351 and P.1 variant was partially (Casirivimab) or fully (Bamlanivimab) resistant to antibodies used for COVID-19 treatment. Moreover, entry of these variants was less efficiently inhibited by plasma from convalescent COVID-19 patients and sera from BNT162b2-vaccinated individuals. These results suggest that SARS-CoV-2 may escape neutralizing antibody responses, which has important implications for efforts to contain the pandemic.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , SARS-CoV-2/inmunología , Animales , COVID-19/inmunología , COVID-19/terapia , COVID-19/virología , Línea Celular , Farmacorresistencia Viral , Humanos , Inmunización Pasiva , Cinética , Fusión de Membrana , Modelos Moleculares , Pruebas de Neutralización , Serina Endopeptidasas/metabolismo , Solubilidad , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunación , Internalización del Virus , Sueroterapia para COVID-19RESUMEN
The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
Asunto(s)
Betacoronavirus/metabolismo , Infecciones por Coronavirus/tratamiento farmacológico , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/tratamiento farmacológico , Inhibidores de Proteasas/farmacología , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus/efectos de los fármacos , Cloruro de Amonio/farmacología , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/química , Betacoronavirus/genética , COVID-19 , Línea Celular , Coronavirus/química , Coronavirus/genética , Coronavirus/fisiología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/terapia , Desarrollo de Medicamentos , Ésteres , Gabexato/análogos & derivados , Gabexato/farmacología , Guanidinas , Humanos , Inmunización Pasiva , Leucina/análogos & derivados , Leucina/farmacología , Pandemias , Peptidil-Dipeptidasa A/química , Receptores Virales/química , Receptores Virales/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Vesiculovirus/genética , Sueroterapia para COVID-19RESUMEN
Hantaviruses are rodent-borne viruses causing serious zoonotic outbreaks worldwide for which no treatment is available. Hantavirus particles are pleomorphic and display a characteristic square surface lattice. The envelope glycoproteins Gn and Gc form heterodimers that further assemble into tetrameric spikes, the lattice building blocks. The glycoproteins, which are the sole targets of neutralizing antibodies, drive virus entry via receptor-mediated endocytosis and endosomal membrane fusion. Here we describe the high-resolution X-ray structures of the heterodimer of Gc and the Gn head and of the homotetrameric Gn base. Docking them into an 11.4-Å-resolution cryoelectron tomography map of the hantavirus surface accounted for the complete extramembrane portion of the viral glycoprotein shell and allowed a detailed description of the surface organization of these pleomorphic virions. Our results, which further revealed a built-in mechanism controlling Gc membrane insertion for fusion, pave the way for immunogen design to protect against pathogenic hantaviruses.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/ultraestructura , Orthohantavirus/química , Glicoproteínas/química , Glicoproteínas/ultraestructura , Orthohantavirus/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiología , Conformación Proteica , Virus ARN , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/ultraestructura , Virión , Internalización del VirusRESUMEN
Arthritogenic alphaviruses, such as Chikungunya virus (CHIKV), cause severe and debilitating rheumatic diseases worldwide, resulting in severe morbidity and economic costs. Recently, MXRA8 was reported as an entry receptor. Here, we present the crystal structures of the mouse MXRA8, human MXRA8 in complex with the CHIKV E protein, and the cryo-electron microscopy structure of human MXRA8 and CHIKV virus-like particle. MXRA8 has two Ig-like domains with unique structural topologies. This receptor binds in the "canyon" between two protomers of the E spike on the surface of the virion. The atomic details at the interface between the two binding entities reveal that both the two domains and the hinge region of MXRA8 are involved in interaction with CHIKV E1-E2 residues from two protomers. Notably, the stalk region of MXRA8 is critical for CHIKV virus entry. This finding provides important information regarding the development of therapeutic countermeasures against those arthritogenic alphaviruses.
Asunto(s)
Virus Chikungunya/química , Proteínas de la Membrana/química , Proteínas del Envoltorio Viral/química , Internalización del Virus , Animales , Virus Chikungunya/metabolismo , Chlorocebus aethiops , Células HEK293 , Humanos , Proteínas de la Membrana/metabolismo , Dominios Proteicos , Células Vero , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Influenza hemagglutinin (HA) is the canonical type I viral envelope glycoprotein and provides a template for the membrane-fusion mechanisms of numerous viruses. The current model of HA-mediated membrane fusion describes a static "spring-loaded" fusion domain (HA2) at neutral pH. Acidic pH triggers a singular irreversible conformational rearrangement in HA2 that fuses viral and cellular membranes. Here, using single-molecule Förster resonance energy transfer (smFRET)-imaging, we directly visualized pH-triggered conformational changes of HA trimers on the viral surface. Our analyses reveal reversible exchange between the pre-fusion and two intermediate conformations of HA2. Acidification of pH and receptor binding shifts the dynamic equilibrium of HA2 in favor of forward progression along the membrane-fusion reaction coordinate. Interaction with the target membrane promotes irreversible transition of HA2 to the post-fusion state. The reversibility of HA2 conformation may protect against transition to the post-fusion state prior to arrival at the target membrane.
Asunto(s)
Membrana Celular/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Virus de la Influenza A/fisiología , Gripe Humana/metabolismo , Imagen Individual de Molécula/métodos , Células A549 , Transferencia Resonante de Energía de Fluorescencia/métodos , Células HEK293 , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Hemaglutininas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Gripe Humana/virología , Unión Proteica , Conformación Proteica , Internalización del VirusRESUMEN
Characterizing cell surface receptors mediating viral infection is critical for understanding viral tropism and developing antiviral therapies. Nevertheless, due to challenges associated with detecting protein interactions on the cell surface, the host receptors of many human pathogens remain unknown. Here, we build a library consisting of most single transmembrane human receptors and implement a workflow for unbiased and high-sensitivity detection of receptor-ligand interactions. We apply this technology to elucidate the long-sought receptor of human cytomegalovirus (HCMV), the leading viral cause of congenital birth defects. We identify neuropilin-2 (Nrp2) as the receptor for HCMV-pentamer infection in epithelial/endothelial cells and uncover additional HCMV interactors. Using a combination of biochemistry, cell-based assays, and electron microscopy, we characterize the pentamer-Nrp2 interaction and determine the architecture of the pentamer-Nrp2 complex. This work represents an important approach to the study of host-pathogen interactions and provides a framework for understanding HCMV infection, neutralization, and the development of novel anti-HCMV therapies.
Asunto(s)
Infecciones por Citomegalovirus/metabolismo , Citomegalovirus/fisiología , Neuropilina-2/metabolismo , Receptores Virales/metabolismo , Anticuerpos Neutralizantes/química , Membrana Celular/metabolismo , Células Endoteliales/metabolismo , Células Epiteliales/metabolismo , Mapeo Epitopo , Femenino , Células HEK293 , Humanos , Conformación Proteica , Proteínas del Envoltorio Viral/metabolismo , Internalización del VirusRESUMEN
How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de la Membrana/química , Membrana Celular/química , Cristalografía por Rayos X , Microscopía Electrónica , Modelos Moleculares , Complejos Multiproteicos/químicaRESUMEN
Sexual reproduction is almost universal in eukaryotic life and involves the fusion of male and female haploid gametes into a diploid cell. The sperm-restricted single-pass transmembrane protein HAP2-GCS1 has been postulated to function in membrane merger. Its presence in the major eukaryotic taxa-animals, plants, and protists (including important human pathogens like Plasmodium)-suggests that many eukaryotic organisms share a common gamete fusion mechanism. Here, we report combined bioinformatic, biochemical, mutational, and X-ray crystallographic studies on the unicellular alga Chlamydomonas reinhardtii HAP2 that reveal homology to class II viral membrane fusion proteins. We further show that targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks gamete fusion. These results demonstrate that HAP2 is the gamete fusogen and suggest a mechanism of action akin to viral fusion, indicating a way to block Plasmodium transmission and highlighting the impact of virus-cell genetic exchanges on the evolution of eukaryotic life.
Asunto(s)
Chlamydomonas/metabolismo , Proteínas de la Fusión de la Membrana/química , Proteínas de Plantas/química , Plasmodium/metabolismo , Proteínas Protozoarias/química , Secuencia de Aminoácidos , Evolución Biológica , Chlamydomonas/citología , Cristalografía por Rayos X , Células Germinativas/química , Células Germinativas/metabolismo , Proteínas de la Fusión de la Membrana/genética , Proteínas de la Fusión de la Membrana/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plasmodium/citología , Dominios Proteicos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
The life cycle of a primary cilium begins in quiescence and ends prior to mitosis. In quiescent cells, the primary cilium insulates itself from contiguous dynamic membrane processes on the cell surface to function as a stable signaling apparatus. Here, we demonstrate that basal restriction of ciliary structure dynamics is established by the cilia-enriched phosphoinositide 5-phosphatase, Inpp5e. Growth induction displaces ciliary Inpp5e and accumulates phosphatidylinositol 4,5-bisphosphate in distal cilia. This change triggers otherwise-forbidden actin polymerization in primary cilia, which excises cilia tips in a process we call cilia decapitation. While cilia disassembly is traditionally thought to occur solely through resorption, we show that an acute loss of IFT-B through cilia decapitation precedes resorption. Finally, we propose that cilia decapitation induces mitogenic signaling and constitutes a molecular link between the cilia life cycle and cell-division cycle. This newly defined ciliary mechanism may find significance in cell proliferation control during normal development and cancer.
Asunto(s)
Ciclo Celular , Cilios/metabolismo , Actinas/metabolismo , Animales , Riñón/citología , Riñón/metabolismo , Ratones , Células 3T3 NIH , Fosfatidilinositol 4,5-Difosfato , Monoéster Fosfórico Hidrolasas/metabolismo , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remodels the endoplasmic reticulum (ER) to form replication organelles, leading to ER stress and unfolded protein response (UPR). However, the role of specific UPR pathways in infection remains unclear. Here, we found that SARS-CoV-2 infection causes marginal activation of signaling sensor IRE1α leading to its phosphorylation, clustering in the form of dense ER-membrane rearrangements with embedded membrane openings, and XBP1 splicing. By investigating the factors regulated by IRE1α-XBP1 during SARS-CoV-2 infection, we identified stress-activated kinase NUAK2 as a novel host-dependency factor for SARS-CoV-2, HCoV-229E, and MERS-CoV entry. Reducing NUAK2 abundance or kinase activity impaired SARS-CoV-2 particle binding and internalization by decreasing cell surface levels of viral receptors and viral trafficking likely by modulating the actin cytoskeleton. IRE1α-dependent NUAK2 levels were elevated in SARS-CoV-2-infected and bystander non-infected cells, promoting viral spread by maintaining ACE2 cell surface levels and facilitating virion binding to bystander cells.
Asunto(s)
Proteínas Serina-Treonina Quinasas , SARS-CoV-2 , Internalización del Virus , Humanos , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP/metabolismo , COVID-19/metabolismo , COVID-19/patología , COVID-19/virología , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , SARS-CoV-2/fisiología , Respuesta de Proteína DesplegadaRESUMEN
The human genome encodes tens of thousands circular RNAs (circRNAs) with mostly unknown functions. Circular RNAs require internal ribosome entry sites (IRES) if they are to undergo translation without a 5' cap. Here, we develop a high-throughput screen to systematically discover RNA sequences that can direct circRNA translation in human cells. We identify more than 17,000 endogenous and synthetic sequences as candidate circRNA IRES. 18S rRNA complementarity and a structured RNA element positioned on the IRES are important for driving circRNA translation. Ribosome profiling and peptidomic analyses show extensive IRES-ribosome association, hundreds of circRNA-encoded proteins with tissue-specific distribution, and antigen presentation. We find that circFGFR1p, a protein encoded by circFGFR1 that is downregulated in cancer, functions as a negative regulator of FGFR1 oncoprotein to suppress cell growth during stress. Systematic identification of circRNA IRES elements may provide important links among circRNA regulation, biological function, and disease.
Asunto(s)
Sitios Internos de Entrada al Ribosoma , Biosíntesis de Proteínas , ARN Circular/metabolismo , Subunidades Ribosómicas/metabolismo , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Conformación de Ácido Nucleico , ARN Circular/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Subunidades Ribosómicas/genética , Relación Estructura-ActividadRESUMEN
Protein-mediated membrane fusion is the dynamic process where specialized protein machinery undergoes dramatic conformational changes that drive two membrane bilayers together, leading to lipid mixing and opening of a fusion pore between previously separate membrane-bound compartments. Membrane fusion is an essential stage of enveloped virus entry that results in viral genome delivery into host cells. Recent studies applying cryo-electron microscopy techniques in a time-resolved fashion provide unprecedented glimpses into the interaction of viral fusion proteins and membranes, revealing fusion intermediate states from the initiation of fusion to release of the viral genome. In combination with complementary structural, biophysical, and computation modeling approaches, these advances are shedding new light on the mechanics and dynamics of protein-mediated membrane fusion.