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
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.
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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
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.
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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
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
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
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.
RESUMEN
Ebola viruses (EBOVs) assemble into filamentous virions, whose shape and stability are determined by the matrix viral protein 40 (VP40). Virus entry into host cells occurs via membrane fusion in late endosomes; however, the mechanism of how the remarkably long virions undergo uncoating, including virion disassembly and nucleocapsid release into the cytosol, remains unknown. Here, we investigate the structural architecture of EBOVs entering host cells and discover that the VP40 matrix disassembles prior to membrane fusion. We reveal that VP40 disassembly is caused by the weakening of VP40-lipid interactions driven by low endosomal pH that equilibrates passively across the viral envelope without a dedicated ion channel. We further show that viral membrane fusion depends on VP40 matrix integrity, and its disassembly reduces the energy barrier for fusion stalk formation. Thus, pH-driven structural remodeling of the VP40 matrix acts as a molecular switch coupling viral matrix uncoating to membrane fusion during EBOV entry.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Humanos , Fiebre Hemorrágica Ebola/metabolismo , Fusión de Membrana , Proteínas del Núcleo Viral/metabolismo , Endosomas/metabolismo , Proteínas de la Matriz ViralRESUMEN
Cellular attachment of viruses determines their cell tropism and species specificity. For entry, vaccinia, the prototypic poxvirus, relies on four binding proteins and an eleven-protein entry fusion complex. The contribution of the individual virus binding proteins to virion binding orientation and membrane fusion is unclear. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards the virus fusion machinery to facilitate fusion. Using a membrane-bleb model system together with super-resolution and electron microscopy we find that side-bound vaccinia virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins reveals that three of four contribute to binding orientation, amongst which the chondroitin sulfate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection. Our results show that viral binding proteins are active participants in successful virus membrane fusion and illustrate the importance of virus protein architecture for successful infection.
Asunto(s)
Poxviridae , Vaccinia , Humanos , Sulfatos de Condroitina , Virus Vaccinia/metabolismo , Poxviridae/metabolismo , Proteínas Virales/metabolismo , Fusión de Membrana , Proteínas PortadorasRESUMEN
A novel pangolin-origin MERS-like coronavirus (CoV), MjHKU4r-CoV-1, was recently identified. It is closely related to bat HKU4-CoV, and is infectious in human organs and transgenic mice. MjHKU4r-CoV-1 uses the dipeptidyl peptidase 4 (DPP4 or CD26) receptor for virus entry and has a broad host tropism. However, the molecular mechanism of its receptor binding and determinants of host range are not yet clear. Herein, we determine the structure of the MjHKU4r-CoV-1 spike (S) protein receptor-binding domain (RBD) complexed with human CD26 (hCD26) to reveal the basis for its receptor binding. Measuring binding capacity toward multiple animal receptors for MjHKU4r-CoV-1, mutagenesis analyses, and homology modeling highlight that residue sites 291, 292, 294, 295, 336, and 344 of CD26 are the crucial host range determinants for MjHKU4r-CoV-1. These results broaden our understanding of this potentially high-risk virus and will help us prepare for possible outbreaks in the future.
Asunto(s)
Dipeptidil Peptidasa 4 , Especificidad del Huésped , Unión Proteica , Receptores Virales , Glicoproteína de la Espiga del Coronavirus , Tropismo Viral , Humanos , Animales , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/química , Dipeptidil Peptidasa 4/metabolismo , Dipeptidil Peptidasa 4/genética , Receptores Virales/metabolismo , Receptores Virales/genética , Receptores Virales/química , Ratones , Sitios de Unión , Internalización del Virus , Modelos Moleculares , Dominios Proteicos , Tropismo al AnfitriónRESUMEN
Severe acute respiratory syndrome virus 2 (SARS-CoV-2) invades host cells by interacting with receptors/coreceptors, as well as with other cofactors, via its spike (S) protein that further mediates fusion between viral and cellular membranes. The host membrane protein, angiotensin-converting enzyme 2 (ACE2), is the major receptor for SARS-CoV-2 and is a crucial determinant for cross-species transmission. In addition, some auxiliary receptors and cofactors are also involved that expand the host/tissue tropism of SARS-CoV-2. After receptor engagement, specific proteases are required that cleave the S protein and trigger its fusogenic activity. Here we discuss the recent advances in understanding the molecular events during SARS-CoV-2 entry which will contribute to developing vaccines and therapeutics.
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COVID-19 , Glicoproteína de la Espiga del Coronavirus , Humanos , Unión Proteica , Receptores Virales/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del VirusRESUMEN
Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments, ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.
Asunto(s)
Anexina A3 , Hepacivirus , Hepatitis C , Antígeno SS-B , Internalización del Virus , Humanos , Anexina A3/metabolismo , Anexina A3/genética , Autoantígenos/metabolismo , Autoantígenos/genética , Células HEK293 , Hepacivirus/metabolismo , Hepacivirus/fisiología , Hepatitis C/metabolismo , Hepatitis C/virología , Hepatitis C/genética , Interacciones Huésped-Patógeno , Gotas Lipídicas/metabolismo , Gotas Lipídicas/virología , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Proteínas del Núcleo Viral/metabolismo , Proteínas del Núcleo Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/genéticaRESUMEN
SARS-CoV-2 is one of the most infectious viruses ever recorded. Despite a plethora of research over the last several years, the viral life cycle is still not well understood, particularly membrane fusion. This process is initiated by the fusion domain (FD), a highly conserved stretch of amino acids consisting of a fusion peptide (FP) and fusion loop (FL), which in synergy perturbs the target cells' lipid membrane to lower the energetic cost necessary for fusion. In this study, through a mutagenesis-based approach, we have investigated the basic residues within the FD (K825, K835, R847, K854) utilizing an in vitro fusion assay and 19F NMR, validated by traditional 13C 15N techniques. Alanine and charge-conserving mutants revealed every basic residue plays a highly specific role within the mechanism of initiating fusion. Intriguingly, K825A led to increased fusogenecity which was found to be correlated to the number of amino acids within helix one, further implicating the role of this specific helix within the FD's fusion mechanism. This work has found basic residues to be important within the FDs fusion mechanism and highlights K825A, a specific mutation made within the FD of the SARS-CoV-2 spike protein, as requiring further investigation due to its potential to contribute to a more virulent strain of SARS-CoV-2.
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Fusión de Membrana , Dominios Proteicos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , Humanos , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/química , COVID-19/virología , COVID-19/metabolismo , Internalización del VirusRESUMEN
SARS-CoV-2 is a newly emerged coronavirus that caused the global COVID-19 outbreak in early 2020. COVID-19 is primarily associated with lung injury, but many other clinical symptoms such as loss of smell and taste demonstrated broad tissue tropism of the virus. Early SARS-CoV-2-host cell interactions and entry mechanisms remain poorly understood. Investigating SARS-CoV-2 infection in tissue culture, we found that the protease TMPRSS2 determines the entry pathway used by the virus. In the presence of TMPRSS2, the proteolytic process of SARS-CoV-2 was completed at the plasma membrane, and the virus rapidly entered the cells within 10 min in a pH-independent manner. When target cells lacked TMPRSS2 expression, the virus was endocytosed and sorted into endolysosomes, from which SARS-CoV-2 entered the cytosol via acid-activated cathepsin L protease 40-60 min post-infection. Overexpression of TMPRSS2 in non-TMPRSS2 expressing cells abolished the dependence of infection on the cathepsin L pathway and restored sensitivity to the TMPRSS2 inhibitors. Together, our results indicate that SARS-CoV-2 infects cells through distinct, mutually exclusive entry routes and highlight the importance of TMPRSS2 for SARS-CoV-2 sorting into either pathway.
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COVID-19/metabolismo , Catepsina L/metabolismo , SARS-CoV-2/fisiología , Serina Endopeptidasas/metabolismo , Animales , COVID-19/genética , Células CACO-2 , Chlorocebus aethiops , Endocitosis , Interacciones Microbiota-Huesped , Humanos , Concentración de Iones de Hidrógeno , Proteolisis , Serina Endopeptidasas/genética , Transducción de Señal , Células Vero , Internalización del VirusRESUMEN
As an intrinsic cellular mechanism responsible for the internalization of extracellular ligands and membrane components, caveolae-mediated endocytosis (CavME) is also exploited by certain pathogens for endocytic entry [e.g., Newcastle disease virus (NDV) of paramyxovirus]. However, the molecular mechanisms of NDV-induced CavME remain poorly understood. Herein, we demonstrate that sialic acid-containing gangliosides, rather than glycoproteins, were utilized by NDV as receptors to initiate the endocytic entry of NDV into HD11 cells. The binding of NDV to gangliosides induced the activation of a non-receptor tyrosine kinase, Src, leading to the phosphorylation of caveolin-1 (Cav1) and dynamin-2 (Dyn2), which contributed to the endocytic entry of NDV. Moreover, an inoculation of cells with NDV-induced actin cytoskeletal rearrangement through Src to facilitate NDV entry via endocytosis and direct fusion with the plasma membrane. Subsequently, unique members of the Rho GTPases family, RhoA and Cdc42, were activated by NDV in a Src-dependent manner. Further analyses revealed that RhoA and Cdc42 regulated the activities of specific effectors, cofilin and myosin regulatory light chain 2, responsible for actin cytoskeleton rearrangement, through diverse intracellular signaling cascades. Taken together, our results suggest that an inoculation of NDV-induced Src-mediated cellular activation by binding to ganglioside receptors. This process orchestrated NDV endocytic entry by modulating the activities of caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPases and downstream effectors. IMPORTANCE: In general, it is known that the paramyxovirus gains access to host cells through direct penetration at the plasma membrane; however, emerging evidence suggests more complex entry mechanisms for paramyxoviruses. The endocytic entry of Newcastle disease virus (NDV), a representative member of the paramyxovirus family, into multiple types of cells has been recently reported. Herein, we demonstrate the binding of NDV to induce ganglioside-activated Src signaling, which is responsible for the endocytic entry of NDV through caveolae-mediated endocytosis. This process involved Src-dependent activation of the caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPase and downstream effectors, thereby orchestrating the endocytic entry process of NDV. Our findings uncover a novel molecular mechanism of endocytic entry of NDV into host cells and provide novel insight into paramyxovirus mechanisms of entry.
Asunto(s)
Macrófagos , Enfermedad de Newcastle , Virus de la Enfermedad de Newcastle , Transducción de Señal , Internalización del Virus , Animales , Endocitosis , Gangliósidos/metabolismo , Macrófagos/metabolismo , Macrófagos/virología , Enfermedad de Newcastle/virología , Virus de la Enfermedad de Newcastle/fisiología , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Coronaviruses (CoVs) pose a major threat to human and animal health worldwide, which complete viral replication by hijacking host factors. Identifying host factors essential for the viral life cycle can deepen our understanding of the mechanisms of virus-host interactions. Based on our previous genome-wide CRISPR screen of α-CoV transmissible gastroenteritis virus (TGEV), we identified the host factor dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), but not DYRK1B, as a critical factor in TGEV replication. Rescue assays and kinase inhibitor experiments revealed that the effect of DYRK1A on viral replication is independent of its kinase activity. Nuclear localization signal modification experiments showed that nuclear DYRK1A facilitated virus replication. Furthermore, DYRK1A knockout significantly downregulated the expression of the TGEV receptor aminopeptidase N (ANPEP) and inhibited viral entry. Notably, we also demonstrated that DYRK1A is essential for the early stage of TGEV replication. Transmission electron microscopy results indicated that DYRK1A contributes to the formation of double-membrane vesicles in a kinase-independent manner. Finally, we validated that DYRK1A is also a proviral factor for mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. In conclusion, our work demonstrated that DYRK1A is an essential host factor for the replication of multiple viruses, providing new insights into the mechanism of virus-host interactions and facilitating the development of new broad-spectrum antiviral drugs.IMPORTANCECoronaviruses, like other positive-sense RNA viruses, can remodel the host membrane to form double-membrane vesicles (DMVs) as their replication organelles. Currently, host factors involved in DMV formation are not well defined. In this study, we used transmissible gastroenteritis virus (TGEV) as a virus model to investigate the regulatory mechanism of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) on coronavirus. Results showed that DYRK1A significantly inhibited TGEV replication in a kinase-independent manner. DYRK1A knockout (KO) can regulate the expression of receptor aminopeptidase N (ANPEP) and endocytic-related genes to inhibit virus entry. More importantly, our results revealed that DYRK1A KO notably inhibited the formation of DMV to regulate the virus replication. Further data proved that DYRK1A is also essential in the replication of mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. Taken together, our findings demonstrated that DYRK1A is a conserved factor for positive-sense RNA viruses and provided new insights into its transcriptional regulation activity, revealing its potential as a candidate target for therapeutic design.
Asunto(s)
Infecciones por Coronavirus , Coronavirus , Quinasas DyrK , Animales , Humanos , Ratones , Antígenos CD13/genética , Coronavirus/clasificación , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Deltacoronavirus , Virus de la Hepatitis Murina/fisiología , Porcinos , Virus de la Gastroenteritis Transmisible/genética , Tirosina , Replicación Viral/fisiología , Quinasas DyrK/metabolismoRESUMEN
Nipah virus (NiV) is a highly lethal, zoonotic Henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor-binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, uses EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor-interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two-point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for the usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, confirm the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV- and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveal regions critical for GhV binding of EFNB2, and describe putative HNV antibody-binding epitopes. IMPORTANCE: Hendra virus and Nipah virus (NiV) are lethal, zoonotic Henipaviruses (HNVs) that cause respiratory and neurological clinical features in humans. Since their initial outbreaks in the 1990s, several novel HNVs have been discovered worldwide, including Ghana virus. Additionally, there is serological evidence of zoonotic transmission, lending way to concerns about future outbreaks. HNV infection of cells is mediated by the receptor-binding protein (RBP) and the Fusion protein (F). The work presented here identifies NiV RBP amino acids important for the usage of ephrin-B3 (EFNB3), a receptor highly expressed in neurons and predicted to be important for neurological clinical features caused by NiV. This study also characterizes epitopes recognized by antibodies against divergent HNV RBPs. Together, this sheds insight to amino acids critical for HNV receptor usage and antibody binding, which is valuable for future studies investigating determinants of viral pathogenesis and developing antibody therapies.
Asunto(s)
Infecciones por Henipavirus , Henipavirus , Receptores Virales , Humanos , Aminoácidos/genética , Anticuerpos Monoclonales/metabolismo , Proteínas Portadoras/metabolismo , Efrina-B3/genética , Efrina-B3/química , Efrina-B3/metabolismo , Epítopos/genética , Epítopos/metabolismo , Ghana , Virus Hendra/metabolismo , Henipavirus/clasificación , Henipavirus/genética , Henipavirus/metabolismo , Mutagénesis , Virus Nipah/metabolismo , Proteínas del Envoltorio Viral/genética , Internalización del Virus , Receptores Virales/metabolismoRESUMEN
Lymphocytic choriomeningitis virus (LCMV) is a rodent-borne zoonotic arenavirus that causes congenital abnormalities and can be fatal for transplant recipients. Using a genome-wide loss-of-function screen, we identify host factors required for LCMV entry into cells. We identify the lysosomal mucin CD164, glycosylation factors, the heparan sulfate biosynthesis machinery, and the known receptor alpha-dystroglycan (α-DG). Biochemical analysis revealed that the LCMV glycoprotein binds CD164 at acidic pH and requires a sialylated glycan at residue N104. We demonstrate that LCMV entry proceeds by the virus switching binding from heparan sulfate or α-DG at the plasma membrane to CD164 prior to membrane fusion, thus identifying additional potential targets for therapeutic intervention.
Asunto(s)
Virus de la Coriomeningitis Linfocítica/fisiología , Internalización del Virus , Células A549 , Sistemas CRISPR-Cas , Endolina/fisiología , Edición Génica , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Concentración de Iones de Hidrógeno , Virus de la Coriomeningitis Linfocítica/patogenicidad , Fusión de Membrana , Factores de VirulenciaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein-catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.