RESUMO
CDHR3 (cadherin-related family member 3) is a transmembrane protein that is highly expressed in airway epithelia and the only known receptor for rhinovirus C (RV-C). A CDHR3 SNP (rs6967330) with G to A base change has been linked to severe exacerbations of asthma and increased susceptibility to RV-C infections in young children. The goals of this study were to determine the subcellular localization of CDHR3 and to test the hypothesis that CDHR3 asthma-risk genotype affects epithelial cell function and susceptibility to RV-C infections of the airway epithelia. We used immunofluorescence imaging, Western blot analysis, and transmission electron microscopy to show CDHR3 subcellular localization in apical cells, including expression in the cilia of airway epithelia. Polymorphisms in CDHR3 rs6967330 locus (GâA) that were previously associated with childhood asthma were related to differences in CDHR3 expression and epithelial cell function. The rs6967330 A allele was associated with higher overall protein expression and RV-C binding and replication compared with the rs6967330 G allele. Furthermore, the rs6967330 A allele was associated with earlier ciliogenesis and higher FOXJ1 expression. Finally, CDHR3 genotype had no significant effects on membrane integrity or ciliary beat function. These findings provide information on the subcellular localization and possible functions of CDHR3 in the airways and link CDHR3 asthma-risk genotype to increased RV-C binding and replication.
Assuntos
Caderinas/genética , Células Epiteliais/virologia , Proteínas de Membrana/genética , Infecções por Picornaviridae/genética , Polimorfismo de Nucleotídeo Único , Mucosa Respiratória/patologia , Rhinovirus/fisiologia , Alelos , Asma/complicações , Asma/genética , Brônquios/patologia , Proteínas Relacionadas a Caderinas , Caderinas/fisiologia , Cílios/química , Células Epiteliais/química , Células Epiteliais/ultraestrutura , Predisposição Genética para Doença , Genótipo , Humanos , Proteínas de Membrana/fisiologia , Infecções por Picornaviridae/virologia , Mucosa Respiratória/virologia , Frações Subcelulares/químicaRESUMO
BACKGROUND: The Rhinovirus C (RV-C), first identified in 2006, produce high symptom burdens in children and asthmatics, however, their primary target host cell in the airways remains unknown. Our primary hypotheses were that RV-C target ciliated airway epithelial cells (AECs), and that cell specificity is determined by restricted and high expression of the only known RV-C cell-entry factor, cadherin related family member 3 (CDHR3). METHODS: RV-C15 (C15) infection in differentiated human bronchial epithelial cell (HBEC) cultures was assessed using immunofluorescent and time-lapse epifluorescent imaging. Morphology of C15-infected differentiated AECs was assessed by immunohistochemistry. RESULTS: C15 produced a scattered pattern of infection, and infected cells were shed from the epithelium. The percentage of cells infected with C15 varied from 1.4 to 14.7% depending on cell culture conditions. Infected cells had increased staining for markers of ciliated cells (acetylated-alpha-tubulin [aat], p < 0.001) but not markers of goblet cells (wheat germ agglutinin or Muc5AC, p = ns). CDHR3 expression was increased on ciliated epithelial cells, but not other epithelial cells (p < 0.01). C15 infection caused a 27.4% reduction of ciliated cells expressing CDHR3 (p < 0.01). During differentiation of AECs, CDHR3 expression progressively increased and correlated with both RV-C binding and replication. CONCLUSIONS: The RV-C only replicate in ciliated AECs in vitro, leading to infected cell shedding. CDHR3 expression positively correlates with RV-C binding and replication, and is largely confined to ciliated AECs. Our data imply that factors regulating differentiation and CDHR3 production may be important determinants of RV-C illness severity.
Assuntos
Brônquios/citologia , Brônquios/virologia , Enterovirus/fisiologia , Células Epiteliais/citologia , Células Epiteliais/virologia , Internalização do Vírus , Replicação Viral/fisiologia , Células Cultivadas , Cílios/fisiologia , Cílios/ultraestrutura , Cílios/virologia , Enterovirus/ultraestrutura , Humanos , Eliminação de Partículas Virais/fisiologiaRESUMO
Rhinoviruses infect ciliated airway epithelial cells, and rhinoviruses' nonstructural proteins quickly inhibit and divert cellular processes for viral replication. However, the epithelium can mount a robust innate antiviral immune response. Therefore, we hypothesized that uninfected cells contribute significantly to the antiviral immune response in the airway epithelium. Using single-cell RNA sequencing, we demonstrate that both infected and uninfected cells upregulate antiviral genes (e.g. MX1, IFIT2, IFIH1, and OAS3) with nearly identical kinetics, whereas uninfected non-ciliated cells are the primary source of proinflammatory chemokines. Furthermore, we identified a subset of highly infectable ciliated epithelial cells with minimal interferon responses and determined that interferon responses originate from distinct subsets of ciliated cells with moderate viral replication. These findings suggest that the composition of ciliated airway epithelial cells and coordinated responses of infected and uninfected cells could determine the risk of more severe viral respiratory illnesses in children with asthma, chronic obstructive pulmonary disease, and genetically susceptible individuals.
Assuntos
Células Epiteliais , Interferons , Criança , Humanos , Células Cultivadas , Imunidade Inata , Expressão Gênica , RhinovirusRESUMO
Here, we assessed the effects of PB1-F2 and NS1 mutations known to increase the pathogenicity of influenza viruses on the replication and pathogenicity in mice of pandemic (H1N1) 2009 influenza viruses. We also characterized viruses possessing a PB1-F2 mutation that was recently identified in pandemic (H1N1) 2009 influenza virus isolates, with and without simultaneous mutations in PB2 and NS1. Our results suggest that some NS1 mutations and the newly identified PB1-F2 mutation have the potential to increase the replication and/or pathogenicity of pandemic (H1N1) 2009 influenza viruses.
Assuntos
Substituição de Aminoácidos/genética , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Animais , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Camundongos , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , VirulênciaRESUMO
Human rhinoviruses (RVs) are picornaviruses that can cause a variety of upper and lower respiratory tract illnesses, including the common cold, bronchitis, pneumonia, and exacerbations of chronic respiratory diseases such as asthma. There are currently > 160 known types of RVs classified into three species (A, B, and C) that use three different cellular membrane glycoproteins expressed in the respiratory epithelium to enter the host cell. These viral receptors are intercellular adhesion molecule 1 (used by the majority of RV-A and all RV-B types), low-density lipoprotein receptor family members (used by 12 RV-A types), and cadherin-related family member 3 (CDHR3; used by RV-C). RV-A and RV-B interactions with intercellular adhesion molecule 1 and low-density lipoprotein receptor glycoproteins are well defined and their cellular functions have been described, whereas the mechanisms of the RV-C interaction with CDHR3 and its cellular functions are being studied. A single nucleotide polymorphism (rs6967330) in CDHR3 increases cell surface expression of this protein and, as a result, also promotes RV-C infections and illnesses. There are currently no approved vaccines or antiviral therapies available to treat or prevent RV infections, which is a major unmet medical need. Understanding interactions between RV and cellular receptors could lead to new insights into the pathogenesis of respiratory illnesses as well as lead to new approaches to control respiratory illnesses caused by RV infections.
Assuntos
Infecções por Picornaviridae , Receptores Virais , Rhinovirus , Interações entre Hospedeiro e Microrganismos , Humanos , Infecções por Picornaviridae/classificação , Infecções por Picornaviridae/imunologia , Infecções por Picornaviridae/virologia , Receptores Virais/classificação , Receptores Virais/fisiologia , Rhinovirus/classificação , Rhinovirus/fisiologiaRESUMO
Viruses in the rhinovirus C species (RV-C) can cause severe respiratory illnesses in children including pneumonia and asthma exacerbations. A transduced cell line (HeLa-E8) stably expressing the CDHR3-Y529 receptor variant, supports propagation of RV-C after infection. C15 clinical or recombinant isolates replicate in HeLa-E8, however progeny yields are lower than those of related strains of RV-A and RV-B. Serial passaging of C15 in HeLa-E8 resulted in stronger cytopathic effects and increased (≥10-fold) virus binding to cells and progeny yields. The adaptation was acquired by two mutations which increased binding (VP1 T125K) and replication (3A E41K), respectively. A similar 3A mutation engineered into C2 and C41 cDNAs also improved viral replication (2-8 fold) in HeLa but the heparan sulfate mediated cell-binding enhancement by the VP1 change was C15-specific. The findings now enable large-scale cost-effective C15 production by infection and the testing of RV-C infectivity by plaque assay.