RESUMO
SARS-CoV-2 entry into host cells is a crucial step for virus tropism, transmission, and pathogenesis. Angiotensin-converting enzyme 2 (ACE2) has been identified as the primary entry receptor for SARS-CoV-2; however, the possible involvement of other cellular components in the viral entry has not yet been fully elucidated. Here we describe the identification of vimentin (VIM), an intermediate filament protein widely expressed in cells of mesenchymal origin, as an important attachment factor for SARS-CoV-2 on human endothelial cells. Using liquid chromatography-tandem mass spectrometry, we identified VIM as a protein that binds to the SARS-CoV-2 spike (S) protein. We showed that the S-protein receptor binding domain (RBD) is sufficient for S-protein interaction with VIM. Further analysis revealed that extracellular VIM binds to SARS-CoV-2 S-protein and facilitates SARS-CoV-2 infection, as determined by entry assays performed with pseudotyped viruses expressing S and with infectious SARS-CoV-2. Coexpression of VIM with ACE2 increased SARS-CoV-2 entry in HEK-293 cells, and shRNA-mediated knockdown of VIM significantly reduced SARS-CoV-2 infection of human endothelial cells. Moreover, incubation of A549 cells expressing ACE2 with purified VIM increased pseudotyped SARS-CoV-2-S entry. CR3022 antibody, which recognizes a distinct epitope on SARS-CoV-2-S-RBD without interfering with the binding of the spike with ACE2, inhibited the binding of VIM with CoV-2 S-RBD, and neutralized viral entry in human endothelial cells, suggesting a key role for VIM in SARS-CoV-2 infection of endothelial cells. This work provides insight into the pathogenesis of COVID-19 linked to the vascular system, with implications for the development of therapeutics and vaccines.
Assuntos
Células Endoteliais/virologia , Espaço Extracelular/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Vimentina/metabolismo , Internalização do Vírus , Células A549 , Enzima de Conversão de Angiotensina 2/metabolismo , Técnicas de Cocultura , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Endotélio Vascular/virologia , Células HEK293 , Humanos , Ligação ProteicaRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1010268.].
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Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness.
Assuntos
Ebolavirus/genética , Infecções por Filoviridae/virologia , Filoviridae/genética , Replicação Viral , Animais , Linhagem Celular , Chlorocebus aethiops , Teste de Complementação Genética , Genoma Viral , Doença pelo Vírus Ebola/virologia , Interações entre Hospedeiro e Microrganismos , Humanos , Corpos de Inclusão/virologia , Células-Tronco Pluripotentes Induzidas/virologia , Macrófagos/virologia , RNA Viral , Genética Reversa , Células Vero , Vírion/genéticaRESUMO
Seasonal influenza carrying key hemagglutinin (HA) head region glycosylation sites can be removed from the lung by pulmonary surfactant protein D (SP-D). Little is known about HA head glycosylation of low-pathogenicity avian influenza virus (LPAIV) subtypes. These can pose a pandemic threat through reassortment and emergence in human populations. Since the presence of head region high-mannose glycosites dictates SP-D activity, the ability to predict these glycosite glycan subtypes may be of value. Here, we investigate the activities of two recombinant human SP-D forms against representative LPAIV strains, including H2N1, H5N1, H6N1, H11N9, an avian H3N8, and a human seasonal H3N2 subtype. Using mass spectrometry, we determined the glycan subclasses and heterogeneities at each head glycosylation site. Sequence alignment and molecular structure analysis of the HAs were performed for LPAIV strains in comparison to seasonal H3N2 and avian H3N8. Intramolecular contacts were determined between the protein backbone and glycosite glycan based on available three-dimensional structure data. We found that glycosite "N165" (H3 numbering) is occupied by high-mannose glycans in H3 HA but by complex glycans in all LPAIV HAs. SP-D was not active on LPAIV but was on H3 HAs. Since SP-D affinity for influenza HA depends on the presence of high-mannose glycan on the head region, our data demonstrate that SP-D may not protect against virus containing these HA subtypes. Our results also demonstrate that glycan subtype can be predicted at some glycosites based on sequence comparisons and three-dimensional structural analysis.IMPORTANCE Low-pathogenicity avian influenza virus (LPAIV) subtypes can reassort with circulating human strains and pandemic viruses can emerge in human populations, as was seen in the 1957 pandemic, in which an H2 virus reassorted with the circulating H1N1 to create a novel H2N2 genotype. Lung surfactant protein D (SP-D), a key factor in first-line innate immunity defense, removes influenza type A virus (IAV) through interaction with hemagglutinin (HA) head region high-mannose glycan(s). While it is known that both H1 and H3 HAs have one or more key high-mannose glycosites in the head region, little is known about similar glycosylation of LPAIV strains H2N1, H5N1, H6N1, or H11N9, which may pose future health risks. Here, we demonstrate that the hemagglutinins of LPAIV strains do not have the required high-mannose glycans and do not interact with SP-D, and that sequence analysis can predict glycan subtype, thus predicting the presence or absence of this virulence marker.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Vírus da Influenza A/metabolismo , Polissacarídeos/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Sequência de Aminoácidos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza A Subtipo H3N8 , Virus da Influenza A Subtipo H5N1 , Modelos Moleculares , Polissacarídeos/química , Conformação Proteica , Análise de Sequência de Proteína , VirulênciaRESUMO
Development of pneumonia is the most lethal consequence of influenza, increasing mortality more than 50-fold compared with uncomplicated infection. The spread of viral infection from conducting airways to the alveolar epithelium is therefore a pivotal event in influenza pathogenesis. We found that mitogenic stimulation with keratinocyte growth factor (KGF) markedly accelerated mortality after infectious challenge with influenza A virus (IAV). Coadministration of KGF with IAV markedly accelerated the spread of viral infection from the airways to alveoli compared with challenge with IAV alone, based on spatial and temporal analyses of viral nucleoprotein staining of lung tissue sections and dissociated lung cells. To better define the temporal relationship between KGF administration and susceptibility to IAV infection in vivo, we administered KGF 120, 48, 24, and 0 h before intrapulmonary IAV challenge and assessed the percentages of proliferating and IAV-infected, alveolar type II (AECII) cells in dispersed lung cell populations. Peak AECII infectivity coincided with the timing of KGF administration that also induced peak AECII proliferation. AECII from mice that were given intrapulmonary KGF before isolation and then infected with IAV ex vivo exhibited the same temporal pattern of proliferation and infectious susceptibility. KGF-induced increases in mortality, AECII proliferation, and enhanced IAV susceptibility were all reversed by pretreatment of the animals with the mTOR inhibitor rapamycin before mitogenic stimulation. Taken together, these data suggest mTOR signaling-dependent, mitogenic conditioning of AECII is a determinant of host susceptibility to infection with IAV.
Assuntos
Células Epiteliais Alveolares/metabolismo , Proliferação de Células/efeitos dos fármacos , Fator 7 de Crescimento de Fibroblastos/farmacologia , Vírus da Influenza A/metabolismo , Mitógenos/farmacologia , Infecções por Orthomyxoviridae/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Suscetibilidade a Doenças/induzido quimicamente , Feminino , Camundongos , Camundongos Endogâmicos DBA , Infecções por Orthomyxoviridae/patologiaRESUMO
Innate immunity is critical in the early containment of influenza A virus (IAV) infection, and surfactant protein D (SP-D) plays a crucial role in the pulmonary defense against IAV. In pigs, which are important intermediate hosts during the generation of pandemic IAVs, SP-D uses its unique carbohydrate recognition domain (CRD) to interact with IAV. An N-linked CRD glycosylation provides interactions with the sialic acid-binding site of IAV, and a tripeptide loop at the lectin-binding site facilitates enhanced interactions with IAV glycans. Here, to investigate both mechanisms of IAV neutralization in greater detail, we produced an N-glycosylated neck-CRD fragment of porcine SP-D (RpNCRD) in HEK293 cells. X-ray crystallography disclosed that the N-glycan did not alter the CRD backbone structure, including the lectin site conformation, but revealed a potential second nonlectin-binding site for glycans. IAV hemagglutination inhibition, IAV aggregation, and neutralization of IAV infection studies showed that RpNCRD, unlike the human analogue RhNCRD, exhibits potent neutralizing activity against pandemic A/Aichi/68 (H3N2), enabled by both porcine-specific structural features of its CRD. MS analysis revealed an N-glycan site-occupancy of >98% at Asn-303 of RpNCRD with complex-type, heterogeneously branched and predominantly α(2,3)-sialylated oligosaccharides. Glycan-binding array data characterized both RpNCRD and RhNCRD as mannose-type lectins. RpNCRD also bound LewisY structures, whereas RhNCRD bound polylactosamine-containing glycans. The presence of the N-glycan in the CRD increases the glycan-binding specificity of RpNCRD. These insights increase our understanding of porcine-specific innate defense against pandemic IAV and may inform the design of recombinant SP-D-based antiviral drugs.
Assuntos
Imunidade Inata/imunologia , Vírus da Influenza A/imunologia , Lectinas/metabolismo , Infecções por Orthomyxoviridae/prevenção & controle , Polissacarídeos/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Ácidos Siálicos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Configuração de Carboidratos , Glicosilação , Testes de Inibição da Hemaglutinação , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Polissacarídeos/química , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/genética , Homologia de Sequência , SuínosRESUMO
Despite sustained biomedical research effort, influenza A virus remains an imminent threat to the world population and a major healthcare burden. The challenge in developing vaccines against influenza is the ability of the virus to mutate rapidly in response to selective immune pressure. Hemagglutinin is the predominant surface glycoprotein and the primary determinant of antigenicity, virulence and zoonotic potential. Mutations leading to changes in the number of HA glycosylation sites are often reported. Such genetic sequencing studies predict at best the disruption or creation of sequons for N-linked glycosylation; they do not reflect actual phenotypic changes in HA structure. Therefore, combined analysis of glycan micro and macro-heterogeneity and bioassays will better define the relationships among glycosylation, viral bioactivity and evolution. We present a study that integrates proteomics, glycomics and glycoproteomics of HA before and after adaptation to innate immune system pressure. We combined this information with glycan array and immune lectin binding data to correlate the phenotypic changes with biological activity. Underprocessed glycoforms predominated at the glycosylation sites found to be involved in viral evolution in response to selection pressures and interactions with innate immune-lectins. To understand the structural basis for site-specific glycan microheterogeneity at these sites, we performed structural modeling and molecular dynamics simulations. We observed that the presence of immature, high-mannose type glycans at a particular site correlated with reduced accessibility to glycan remodeling enzymes. Further, the high mannose glycans at sites implicated in immune lectin recognition were predicted to be capable of forming trimeric interactions with the immune-lectin surfactant protein-D.
Assuntos
Glicômica/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/metabolismo , Polissacarídeos/análise , Proteômica/métodos , Biologia Computacional/métodos , Cristalografia por Raios X , Glicosilação , Humanos , Imunidade Inata , Vírus da Influenza A/química , Manose/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Polissacarídeos/químicaRESUMO
We recently reported that a trimeric neck and carbohydrate recognition domain (NCRD) fragment of human surfactant protein D (SP-D), a host defense lectin, with combinatorial substitutions at the 325 and 343 positions (D325A+R343V) exhibits markedly increased antiviral activity for seasonal strains of influenza A virus (IAV). The NCRD binds to glycan-rich viral envelope proteins including hemagglutinin (HA). We now show that replacement of D325 with serine to create D325S+R343V provided equal or increased neutralizing activity compared with D325A+R343V. The activity of the double mutants was significantly greater than that of either single mutant (D325A/S or R343V). D325A+R343V and D325S+R343V also strongly inhibited HA activity, and markedly aggregated, the 1968 pandemic H3N2 strain, Aichi68. D325S+R343V significantly reduced viral loads and mortality of mice infected with Aichi68, whereas wild-type SP-D NCRD did not. The pandemic H1N1 strains of 1918 and 2009 have only one N-linked glycan side on the head region of the HA and are fully resistant to inhibition by native SP-D. Importantly, we now show that D325A+R343V and D325S+R343V inhibited Cal09 H1N1 and related strains, and reduced uptake of Cal09 by epithelial cells. Inhibition of Cal09 was mediated by the lectin activity of the NCRDs. All known human pandemic strains have at least one glycan attachment on the top or side of the HA head, and our results indicate that they may be susceptible to inhibition by modified host defense lectins.
Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/virologia , Proteína D Associada a Surfactante Pulmonar/genética , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Resistência à Doença , Cães , Feminino , Interações Hospedeiro-Patógeno , Humanos , Influenza Humana/imunologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos DBA , Mutação de Sentido Incorreto , Pandemias , Ligação Proteica , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/metabolismo , Carga ViralRESUMO
The collectins have been shown to have a role in host defense against influenza A virus (IAV) and other significant viral pathogens (e.g., HIV). The ficolins are a related group of innate immune proteins that are present at relatively high concentrations in serum, but also in respiratory secretions; however, there has been little study of the role of ficolins in viral infection. In this study, we demonstrate that purified recombinant human H-ficolin and H-ficolin in human serum and bronchoalveolar lavage fluid bind to IAV and inhibit viral infectivity and hemagglutination activity in vitro. Removal of ficolins from human serum or bronchoalveolar lavage fluid reduces their antiviral activity. Inhibition of IAV did not involve the calcium-dependent lectin activity of H-ficolin. We demonstrate that H-ficolin is sialylated and that removal of sialic acid abrogates IAV inhibition, while addition of the neuraminidase inhibitor oseltamivir potentiates neutralization, hemagglutinin inhibition, and viral aggregation caused by H-ficolin. Pandemic and mouse-adapted strains of IAV are generally not inhibited by the collectins surfactant protein D or mannose binding lectin because of a paucity of glycan attachments on the hemagglutinin of these strains. In contrast, H-ficolin inhibited both the mouse-adapted PR-8 H1N1 strain and a pandemic H1N1 strain from 2009. H-ficolin also fixed complement to a surface coated with IAV. These findings suggest that H-ficolin contributes to host defense against IAV.
Assuntos
Antivirais/farmacologia , Glicoproteínas/fisiologia , Vírus da Influenza A Subtipo H3N2/imunologia , Lectinas/fisiologia , Pandemias , Estações do Ano , Replicação Viral/imunologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Testes de Fixação de Complemento , Cães , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H3N2/patogenicidade , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Camundongos , Testes de Neutralização , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Proteínas da Matriz Viral/antagonistas & inibidoresRESUMO
Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus-host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results.
RESUMO
Surfactant protein D (SP-D), a mammalian C-type lectin, is the primary innate inhibitor of influenza A virus (IAV) in the lung. Interactions of SP-D with highly branched viral N-linked glycans on hemagglutinin (HA), an abundant IAV envelope protein and critical virulence factor, promote viral aggregation and neutralization through as yet unknown molecular mechanisms. Two truncated human SP-D forms, wild-type (WT) and double mutant D325A+R343V, representing neck and carbohydrate recognition domains are compared in this study. Whereas both WT and D325A+R343V bind to isolated glycosylated HA, WT does not inhibit IAV in neutralization assays; in contrast, D325A+R343V neutralization compares well with that of full-length native SP-D. To elucidate the mechanism for these biochemical observations, we have determined crystal structures of D325A+R343V in the presence and absence of a viral nonamannoside (Man9). On the basis of the D325A+R343V-Man9 structure and other crystallographic data, models of complexes between HA and WT or D325A+R343V were produced and subjected to molecular dynamics. Simulations reveal that whereas WT and D325A+R343V both block the sialic acid receptor site of HA, the D325A+R343V complex is more stable, with stronger binding caused by additional hydrogen bonds and hydrophobic interactions with HA residues. Furthermore, the blocking mechanism of HA differs for WT and D325A+R343V because of alternate glycan binding modes. The combined results suggest a mechanism through which the mode of SP-D-HA interaction could significantly influence viral aggregation and neutralization. These studies provide the first atomic-level molecular view of an innate host defense lectin inhibiting its viral glycoprotein target.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Modelos Moleculares , Proteína D Associada a Surfactante Pulmonar/química , Adesividade , Substituição de Aminoácidos , Sítios de Ligação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Vírus da Influenza A Subtipo H3N2/química , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H3N2/metabolismo , Viabilidade Microbiana , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Conformação Proteica , Proteína D Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Pigs can act as intermediate hosts by which reassorted influenza A virus (IAV) strains can be transmitted to humans and cause pandemic influenza outbreaks. The innate host defense component surfactant protein D (SP-D) interacts with glycans on the hemagglutinin of IAV and contributes to protection against IAV infection in mammals. This study shows that a recombinant trimeric neck lectin fragment derived from porcine SP-D (pSP-D) exhibits profound inhibitory activity against IAV, in contrast to comparable fragments derived from human SP-D. Crystallographic analysis of the pSP-D fragment complexed with a viral sugar component shows that a unique tripeptide loop alters the lectin site conformation of pSP-D. Molecular dynamics simulations highlight the role of this flexible loop, which adopts a more stable conformation upon sugar binding and may facilitate binding to viral glycans through contact with distal portions of the branched mannoside. The combined data demonstrate that porcine-specific structural features of SP-D contribute significantly to its distinct anti-IAV activity. These findings could help explain why pigs serve as important reservoirs for newly emerging pathogenic IAV strains.
Assuntos
Antivirais/farmacologia , Metabolismo dos Carboidratos , Vírus da Influenza A/efeitos dos fármacos , Proteína D Associada a Surfactante Pulmonar/farmacologia , Animais , Antivirais/química , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Cristalização , Primers do DNA , Cães , Testes de Sensibilidade Microbiana , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/genética , SuínosRESUMO
LL-37, the only human cathelicidin, is a cationic antimicrobial peptide with antibacterial and antifungal activity. LL-37 is released from neutrophil granules and produced by epithelial cells. It has been implicated in host defence against influenza A virus (IAV) in recent studies. We now demonstrate dose-related neutralizing activity of LL-37 against several seasonal and mouse-adapted IAV strains. The ability of LL-37 to inhibit these IAV strains resulted mainly from direct effects on the virus, since pre-incubation of virus with LL-37 was needed for optimal inhibition. LL-37 bound high-density lipoprotein (HDL), and pre-incubation of LL-37 with human serum or HDL reduced its antiviral activity. LL-37 did not inhibit viral association with epithelial cells as assessed by quantitative RT-PCR or confocal microscopy. This finding contrasted with results obtained with surfactant protein D (SP-D). Unlike collectins or human neutrophil defensins (HNPs), LL-37 did not induce viral aggregation under electron microscopy. In the electron microscopy studies, LL-37 appeared to cause disruption of viral membranes. LL-37 had additive antiviral activity when combined with other innate inhibitors like SP-D, surfactant protein A and HNPs. Unlike HNPs, LL-37 did not bind SP-D significantly. These findings indicate that LL-37 contributes to host defence against IAV through a mechanism distinct from that of SP-D and HNPs.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Defensinas/metabolismo , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/metabolismo , Vírus da Influenza A/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Peptídeos Catiônicos Antimicrobianos/imunologia , Células CHO , Colectinas/imunologia , Colectinas/metabolismo , Cricetinae , Cricetulus , Defensinas/imunologia , Cães , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A/imunologia , Lipoproteínas HDL/imunologia , Lipoproteínas HDL/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/virologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Proteína A Associada a Surfactante Pulmonar/imunologia , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/imunologia , CatelicidinasRESUMO
The study of highly pathogenic viruses handled under BSL-4 conditions and classified as Select Agents frequently involves the transfer of inactivated materials to lower containment levels for downstream analyses. Adhering to Select Agent and BSL-4 safety regulations requires validation or verification of the inactivation procedures, which comes with an array of challenges for each method. This includes the use of cytotoxic reagents for chemical inactivation and defining the precise inactivation parameters for physical inactivation. Here, we provide a workflow for various inactivation methods using Ebola, Nipah, and Lassa viruses as our examples. We choose three distinct inactivation methods (TRIzol/TRIzol LS, aldehyde fixation using different fixatives, and heat) to highlight the challenges of each method and provide possible solutions. We show that, whereas published chemical inactivation methods are highly reliable, the parameters for heat inactivation must be clearly defined to ensure complete inactivation. In addition to the inactivation data, we also provide examples and templates for the documentation required for approval and use of inactivation SOPs, including an inactivation report, the procedure sections of developed SOPs, and an electronic inactivation certificate that accompanies inactivated samples. The provided information can be used as a roadmap for similar studies at high and maximum containment laboratories.
RESUMO
Cardiovascular complications are major clinical hallmarks of acute and post-acute coronavirus disease 2019 (COVID-19). However, the mechanistic details of SARS-CoV-2 infectivity of endothelial cells remain largely unknown. Here, we demonstrate that the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein shares a similarity with the proline-rich binding ena/VASP homology (EVH1) domain and identified the endoplasmic reticulum (ER) resident calreticulin (CALR) as an S-RBD interacting protein. Our biochemical analysis showed that CALR, via its proline-rich (P) domain, interacts with S-RBD and modulates proteostasis of the S protein. Treatment of cells with the proteasomal inhibitor bortezomib increased the expression of the S protein independent of CALR, whereas the lysosomal/autophagy inhibitor bafilomycin 1A, which interferes with the acidification of lysosome, selectively augmented the S protein levels in a CALR-dependent manner. More importantly, the shRNA-mediated knockdown of CALR increased SARS-CoV-2 infection and impaired calcium homeostasis of human endothelial cells. This study provides new insight into the infectivity of SARS-CoV-2, calcium hemostasis, and the role of CALR in the ER-lysosome-dependent proteolysis of the spike protein, which could be associated with cardiovascular complications in COVID-19 patients.
Assuntos
Calreticulina , Síndrome de COVID-19 Pós-Aguda , Humanos , Cálcio/metabolismo , Calreticulina/metabolismo , Células Endoteliais/metabolismo , Prolina , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Síndrome de COVID-19 Pós-Aguda/metabolismoRESUMO
Porcine surfactant protein D (pSP-D) displays distinctively strong, broad-range inhibitory activity against influenza A virus (IAV). N-Linked glycosylation of the carbohydrate recognition domain (CRD) of pSP-D contributes to the high affinity of this collectin for IAV. To investigate the role of the N-linked glycan further, HEK293E protein expression was used to produce recombinant pSP-D (RpSP-D) that has similar structural and antiviral properties as NpSP-D. We introduced an additional N-linked glycan in the CRD of RpSP-D but this modification did not alter the antiviral activity. Human SP-D is unglycosylated in its CRD and less active against IAV compared with pSP-D. In an attempt to modify its antiviral properties, several recombinant human SP-D (RhSP-D) mutants were constructed with N-linked glycans introduced at various locations within its CRD. To retain lectin activity, necessary for the primary interactions between SP-D and IAV, N-linked glycosylation of RhSP-D was shown to be restricted to the corresponding position in the CRD of either pSP-D or surfactant protein A (SP-A). These N-glycosylated RhSP-D mutants, however, did not show increased neutralization activity against IAV. By developing RhSP-D mutants that also have the pSP-D-specific Ser-Gly-Ala loop inserted in the CRD, we could demonstrate that the N-linked glycan-mediated interactions between pSP-D and IAV involves additional structural prerequisites of the pSP-D CRD. Ultimately, these studies will help to develop highly effective SP-D-based therapeutic and prophylactic drugs against IAV.
Assuntos
Vírus da Influenza A/metabolismo , Lectinas , Polissacarídeos/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Animais , Cães , Glicosilação , Células HEK293 , Humanos , Vírus da Influenza A/química , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/terapia , Mutação , Polissacarídeos/química , Polissacarídeos/genética , Proteína A Associada a Surfactante Pulmonar/química , Proteína A Associada a Surfactante Pulmonar/genética , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SuínosRESUMO
The recognition of influenza A virus (IAV) by surfactant protein D (SP-D) is mediated by interactions between the SP-D carbohydrate recognition domains (CRD) and glycans displayed on envelope glycoproteins. Although native human SP-D shows potent antiviral and aggregating activity, trimeric recombinant neck+CRDs (NCRDs) show little or no capacity to influence IAV infection. A mutant trimeric NCRD, D325A/R343V, showed marked hemagglutination inhibition and viral neutralization, with viral aggregation and aggregation-dependent viral uptake by neutrophils. D325A/R343V exhibited glucose-sensitive binding to Phil82 hemagglutinin trimer (HA) by surface plasmon resonance. By contrast, there was very low binding to the HA trimer from another virus (PR8) that lacks glycans on the HA head. Mass spectrometry demonstrated the presence of high mannose glycans on the Phil82 HA at positions known to contribute to IAV binding. Molecular modeling predicted an enhanced capacity for bridging interactions between HA glycans and D325A/R343V. Finally, the trimeric D325A/R343V NCRD decreased morbidity and increased viral clearance in a murine model of IAV infection using a reassortant A/WSN/33 virus with a more heavily glycosylated HA. The combined data support a model in which altered binding by a truncated mutant SP-D to IAV HA glycans facilitates viral aggregation, leading to significant viral neutralization in vitro and in vivo. These studies demonstrate the potential utility of homology modeling and protein structure analysis for engineering effective collectin antivirals as in vivo therapeutics.
Assuntos
Resistência à Doença/genética , Evolução Molecular , Vírus da Influenza A Subtipo H1N1/fisiologia , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/genética , Ressonância de Plasmônio de Superfície/métodos , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Cristalografia por Raios X , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/metabolismo , Espectrometria de Massas , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Infecções por Orthomyxoviridae/virologia , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteína D Associada a Surfactante Pulmonar/metabolismo , Especificidade da EspécieRESUMO
Mannose-binding lectin (MBL), a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid ß peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to Aß are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Inflamação/metabolismo , Lectina de Ligação a Manose/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Animais , Cálcio/metabolismo , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Humanos , Inflamação/imunologia , Inflamação/microbiologia , Vírus da Influenza A/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Polissacarídeos/metabolismo , Polissacarídeos/farmacologia , Staphylococcus aureus/imunologiaRESUMO
BACKGROUND: Excessive inflammatory host response increases morbidity and mortality associated with seasonal respiratory influenza, and highly pathogenic virus strains are characterized by massive infiltration of monocytes and/or macrophages that produce a storm of injurious cytokines. METHODS: Here, we examined the role in respiratory influenza of serpinB1, an endogenous inhibitor of the serine proteases elastase, cathepsin G, and proteinase-3, increasingly recognized as regulators of inflammation. RESULTS: After challenge with high-dose surfactant protein-D (SP-D)-sensitive influenza A/Philadelphia/82 (H3N2), serpinB1(-/-) mice died earlier and in greater numbers than did wild-type mice. Sublethally infected animals suffered increased morbidity, delayed resolution of epithelial injury, and increased immune cell death. Viral clearance and SP-D/SP-A upregulation were unimpaired and so were early virus-induced cytokine and chemokine burst and influx of large numbers of neutrophils and monocytes. Whereas initial cytokines and chemokines rapidly cleared in wild-type mice, TNF-α, IL-6, KC/CXCL1, G-CSF, IL-17A, and MCP-1/CCL2 remained elevated in serpinB1(-/-) mice. Monocyte-derived cells were the dominant immune cells in influenza-infected lungs, and those from serpinB1(-/-) mice produced excessive IL-6 and TNF-α when tested ex vivo. Pulmonary γδ T-cells that produced IL-17A were also increased. CONCLUSIONS: Because viral clearance was unimpaired, the study highlights the critical role of serpinB1 in mitigating inflammation and restricting pro-inflammatory cytokine production in influenza infection.
Assuntos
Inflamação/metabolismo , Pneumopatias/virologia , Pulmão/patologia , Infecções por Orthomyxoviridae/patologia , Serpinas/metabolismo , Animais , Morte Celular , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/fisiologia , Inflamação/patologia , Vírus da Influenza A Subtipo H3N2 , Pneumopatias/patologia , Camundongos , Camundongos Knockout , Infecções por Orthomyxoviridae/imunologia , Serpinas/genética , Fatores de TempoRESUMO
We have reported that the alpha-defensins human neutrophil peptides (HNP)-1 and HNP-2 neutralize and aggregate influenza A virus (IAV) and promote uptake of IAV by neutrophils. These alpha-defensins were also shown to bind to surfactant protein (SP)-D and reduce its antiviral activity. In this study, we examined retrocyclin (RC)1 and RC2, humanized versions of the antiviral theta-defensins found in the leukocytes of certain nonhuman primates. RC1 was just as effective as HNP-1-3 in neutralizing IAV, and RC2 and RC101 (an analog of RC1) were more effective. In contrast, human beta-defensins (HBDs) showed less neutralizing activity. Human defensins 5 and 6 (mainly produced by intestinal Paneth cells) had viral neutralizing activity similar to HNP-1-3. Like HNP-1-3, RCs induced viral aggregation and promoted the uptake of IAV by neutrophils. We used surface plasmon resonance to evaluate binding of defensins to SP-D. HBDs, HD6, and HNP-4 bound minimally to SP-D. HNP-1-3 and RCs bound SP-D with high affinity; however, unlike HNP-1 and HNP-2, RCs did not inhibit SP-D antiviral activity. HBDs also did not inhibit antiviral activity of SP-D. Given their strong neutralizing activity and compatibility with SP-D, RCs may provide attractive prototypes for designing therapeutics that can prevent or treat respiratory infections caused by IAV.