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As intracellular parasites, viruses exploit cellular proteins at every stage of infection. Adenovirus outbreaks are associated with severe acute respiratory illnesses and conjunctivitis, with no specific antiviral therapy available. An adenoviral vaccine based on human adenovirus species D (HAdV-D) is currently in use for COVID-19. Herein, we investigate host interactions of HAdV-D type 37 (HAdV-D37) protein IIIa (pIIIa), identified by affinity purification and mass spectrometry (AP-MS) screens. We demonstrate that viral pIIIa interacts with ubiquitin-specific protease 9x (USP9x) and Ran-binding protein 2 (RANBP2). USP9x binding did not invoke its signature deubiquitination function but rather deregulated pIIIa-RANBP2 interactions. In USP9x-knockout cells, viral genome replication and viral protein expression increased compared to wild type cells, supporting a host-favored mechanism for USP9x. Conversely, RANBP2-knock down reduced pIIIa transport to the nucleus, viral genome replication, and viral protein expression. Also, RANBP2-siRNA pretreated cells appeared to contain fewer mature viral particles. Transmission electron microscopy of USP9x-siRNA pretreated, virus-infected cells revealed larger than typical paracrystalline viral arrays. RANBP2-siRNA pretreatment led to the accumulation of defective assembly products at an early maturation stage. CRM1 nuclear export blockade by leptomycin B led to the retention of pIIIa within cell nuclei and hindered pIIIa-RANBP2 interactions. In-vitro binding analyses indicated that USP9x and RANBP2 bind to C-terminus of pIIIa amino acids 386-563 and 386-510, respectively. Surface plasmon resonance testing showed direct pIIIa interaction with recombinant USP9x and RANBP2 proteins, without competition. Using an alternative and genetically disparate adenovirus type (HAdV-C5), we show that the demonstrated pIIIa interaction is also important for a severe respiratory pathogen. Together, our results suggest that pIIIa hijacks RANBP2 for nuclear import and subsequent virion assembly. USP9x counteracts this interaction and negatively regulates virion synthesis. This analysis extends the scope of known adenovirus-host interactions and has potential implications in designing new antiviral therapeutics.
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Infecções por Adenoviridae , Adenovírus Humanos , COVID-19 , Transporte Ativo do Núcleo Celular , Adenoviridae/genética , Adenovírus Humanos/genética , Humanos , Chaperonas Moleculares , Complexo de Proteínas Formadoras de Poros Nucleares , RNA Interferente Pequeno , Ubiquitina Tiolesterase/genética , Proteases Específicas de Ubiquitina , Proteínas Virais/genéticaRESUMO
With the advent of high-resolution and cost-effective genomics and bioinformatics tools and methods contributing to a large database of both human (HAdV) and simian (SAdV) adenoviruses, a genomics-based re-evaluation of their taxonomy is warranted. Interest in these particular adenoviruses is growing in part due to the applications of both in gene transfer protocols, including gene therapy and vaccines, as well in oncolytic protocols. In particular, the re-evaluation of SAdVs as appropriate vectors in humans is important as zoonosis precludes the assumption that human immune system may be naïve to these vectors. Additionally, as important pathogens, adenoviruses are a model organism system for understanding viral pathogen emergence through zoonosis and anthroponosis, particularly among the primate species, along with recombination, host adaptation, and selection, as evidenced by one long-standing human respiratory pathogen HAdV-4 and a recent re-evaluation of another, HAdV-76. The latter reflects the insights on amphizoonosis, defined as infections in both directions among host species including "other than human", that are possible with the growing database of nonhuman adenovirus genomes. HAdV-76 is a recombinant that has been isolated from human, chimpanzee, and bonobo hosts. On-going and potential impacts of adenoviruses on public health and translational medicine drive this evaluation of 174 whole genome sequences from HAdVs and SAdVs archived in GenBank. The conclusion is that rather than separate HAdV and SAdV phylogenetic lineages, a single, intertwined tree is observed with all HAdVs and SAdVs forming mixed clades. Therefore, a single designation of "primate adenovirus" (PrAdV) superseding either HAdV and SAdV is proposed, or alternatively, keeping HAdV for human adenovirus but expanding the SAdV nomenclature officially to include host species identification as in ChAdV for chimpanzee adenovirus, GoAdV for gorilla adenovirus, BoAdV for bonobo adenovirus, and ad libitum.
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Adenovírus Humanos/genética , Adenovirus dos Símios/genética , Genoma Viral , Infecções por Adenoviridae , Adenovírus Humanos/classificação , Adenovirus dos Símios/classificação , Animais , Evolução Molecular , Genômica , Humanos , Filogenia , ZoonosesRESUMO
Interleukin-1ß (IL-1ß) is a potent mediator of innate immunity commonly up-regulated in a broad spectrum of inflammatory diseases. When bound to its cell surface receptor, IL-1ß initiates a signalling cascade that cooperatively induces the expression of canonical IL-1 target genes such as IL-8 and IL-6. Here, we present galectin-3 as a novel regulator of IL-1ß responses in corneal keratinocytes. Using the SNAP-tag system and digitonin semi-permeabilization, we show that recombinant exogenous galectin-3 binds to the plasma membrane of keratinocytes and is internalized into cytoplasmic compartments. We find that exogenous galectin-3, but not a dominant negative inhibitor of galectin-3 polymerization lacking the N-terminal domain, exacerbates the response to IL-1ß by stimulating the secretion of inflammatory cytokines. The activity of galectin-3 could be reduced by a novel d-galactopyranoside derivative targeting the conserved galactoside-binding site of galectins and did not involve interaction with IL-1 receptor 1 or the induction of endogenous IL-1ß. Consistent with these observations, we demonstrate that small interfering RNA-mediated suppression of endogenous galectin-3 expression is sufficient to impair the IL-1ß-induced secretion of IL-8 and IL-6 in a p38 mitogen-activated protein kinase-independent manner. Collectively, our findings provide a novel role for galectin-3 as an amplifier of IL-1ß responses during epithelial inflammation through an as yet unidentified mechanism.
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Galectina 3/metabolismo , Interleucina-1beta/metabolismo , Queratinócitos/metabolismo , Ceratite/etiologia , Ceratite/metabolismo , Células Cultivadas , Endocitose , Galectina 3/farmacologia , Humanos , Interleucina-1beta/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Ceratite/patologia , Ligação ProteicaRESUMO
Pattern recognition receptors (PRRs) are critical to the early detection and innate immune responses to pathogens. In particular, the toll-like receptor (TLR) system and its associated adaptor proteins have essential roles in early host responses to infection. Epidemic keratoconjunctivitis, caused by the human adenovirus, is a severe ocular surface infection associated with corneal inflammation (stromal keratitis). We previously showed that adenovirus capsid was a key molecular pattern in adenovirus keratitis, with viral DNA having a lesser role. We have now investigated the role of the adaptor molecule MyD88 in a mouse model of adenovirus keratitis in which there is no viral replication. In MyD88-/- mice infected with human adenovirus type 37, clinical keratitis was markedly reduced, along with infiltration of CD45+ cells, and expression of inflammatory cytokines. Reduction of inflammatory cytokines was also observed in infected primary human corneal fibroblasts pretreated with a MyD88 inhibitory peptide. Keratitis similar to wild type mice was observed in TLR2, TLR9 and IL-1R knockout mice, but was reduced in TLR2/9 double knockout mice, consistent with synergy of TLR2 and TLR9 in the response to adenovirus infection. MyD88 co-immunoprecipitated with Src kinase in mice corneas and in human corneal fibroblasts infected with adenovirus, and MyD88 inhibitory peptide reduced Src phosphorylation, linking MyD88 activation to inflammatory gene expression through a signaling cascade previously shown to be directed by Src. Our findings reveal a critical role for the PRRs TLR2 and 9, and their adaptor protein MyD88, in corneal inflammation upon adenovirus infection.
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Infecções por Adenoviridae/metabolismo , Infecções por Adenoviridae/patologia , Adenoviridae/fisiologia , Ceratite/metabolismo , Ceratite/virologia , Fator 88 de Diferenciação Mieloide/metabolismo , Células A549 , Adenoviridae/genética , Infecções por Adenoviridae/virologia , Animais , Córnea/patologia , Córnea/virologia , Feminino , Humanos , Ceratite/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-1/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Internalização do Vírus , Quinases da Família src/metabolismoRESUMO
Human adenoviruses (HAdVs) contain seven species (HAdV-A to -G), each associated with specific disease conditions. Among these, HAdV-D includes those viruses associated with epidemic keratoconjunctivitis (EKC), a severe ocular surface infection. The reasons for corneal tropism for some but not all HAdV-Ds are not known. The fiber protein is a major capsid protein; its C-terminal "knob" mediates binding with host cell receptors to facilitate subsequent viral entry. In a comprehensive phylogenetic analysis of HAdV-D capsid genes, fiber knob gene sequences of HAdV-D types associated with EKC formed a unique clade. By proteotyping analysis, EKC virus-associated fiber knobs were uniquely shared. Comparative structural modeling showed no distinct variations in fiber knobs of EKC types but did show variation among HAdV-Ds in a region overlapping with the known CD46 binding site in HAdV-B. We also found signature amino acid positions that distinguish EKC from non-EKC types, and by in vitro studies we showed that corneal epithelial cell tropism can be predicted by the presence of a lysine or alanine at residue 240. This same amino acid residue in EKC viruses shows evidence for positive selection, suggesting that evolutionary pressure enhances fitness in corneal infection, and may be a molecular determinant in EKC pathogenesis. IMPORTANCE: Viruses adapt various survival strategies to gain entry into target host cells. Human adenovirus (HAdV) types are associated with distinct disease conditions, yet evidence for connections between genotype and cellular tropism is generally lacking. Here, we provide a structural and evolutionary basis for the association between specific genotypes within HAdV species D and epidemic keratoconjunctivitis, a severe ocular surface infection. We find that HAdV-D fiber genes of major EKC pathogens, specifically the fiber knob gene region, share a distinct phylogenetic clade. Deeper analysis of the fiber gene revealed that evolutionary pressure at crucial amino acid sites has a significant impact on its structural conformation, which is likely important in host cell binding and entry. Specific amino acids in hot spot residues provide a link to ocular cell tropism and possibly to corneal pathogenesis.
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Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/genética , Ceratoconjuntivite/virologia , Células A549 , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , Linhagem Celular Tumoral , Córnea/virologia , DNA Viral/genética , Genótipo , Humanos , Filogenia , Alinhamento de Sequência/métodos , Análise de Sequência de DNA , Internalização do VírusRESUMO
Activation of protein kinase C (PKC), a serine/threonine protein kinase, ubiquitously influences cellular signal transduction and has been shown to play a role in viral entry. In this study, we explored a role for PKC in human adenovirus type 37 infection of primary human corneal fibroblasts, a major target cell for infection. We sought evidence for an interaction between PKC activation and two potential downstream targets: cSrc kinase, shown previously to play a critical role in adenovirus signaling in these cells, and caveolin-1, reported earlier to be important to entry of adenovirus type 37. Infection of fibroblasts increased PKCα phosphorylation and translocation of PKCα from the cytosol to caveolin-1 containing vesicles. Virus-induced phosphorylation of both cSrc and AKT was abolished in cell lysates pretreated with calphostin C, a chemical inhibitor of PKC. Inhibition of PKC also reduced virus associated phosphorylation of caveolin-1, while inhibition of cSrc by the chemical inhibitor PP2 reduced only caveolin-1 phosphorylation, but not PKCα phosphorylation, in lipid rafts. These results suggest a role for PKCα upstream to both cSrc and caveolin-1. Phosphorylated PKCα was found in the same endosomal fractions as phosphorylated cSrc, and PKCα was present to a greater degree in caveolin-1 pull downs from virus infected than mock infected cell lysates. Calphostin C also reduced early viral gene expression, indicating that PKCα activity may be required for viral entry. PKCα plays a central role in adenovirus infection of corneal fibroblasts and regulation of downstream molecules, including the important lipid raft component caveolin-1.
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The cornea contains a heterogeneous population of antigen-presenting cells with the capacity to contribute to immune responses. Adenovirus keratitis is a severe corneal infection with acute and chronic phases. The role of resident corneal antigen-presenting cells in adenovirus keratitis has not been studied. We utilized transgenic MaFIA mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, in a mouse model of adenovirus keratitis. Clinical keratitis and recruitment of myeloperoxidase and CD45(+) cells were diminished in c-fms depleted, adenovirus infected mice, as compared to controls, consistent with a role for myeloid-lineage cells in adenovirus keratitis.
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Infecções por Adenoviridae/patologia , Células Dendríticas/citologia , Infecções Oculares Virais/patologia , Ceratite/patologia , Macrófagos/citologia , Infecções por Adenoviridae/imunologia , Animais , Quimiocina CXCL1/metabolismo , Córnea/citologia , Córnea/imunologia , Substância Própria/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Infecções Oculares Virais/imunologia , Infecções Oculares Virais/virologia , Ceratite/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismoRESUMO
Purpose: To develop a 3-dimensional corneal construct suitable for in vitro studies of disease conditions and therapies. Design: In vitro human corneal constructs were created using chemically crosslinked collagen and chondroitin sulfate extracellular matrix and seeded with 3 human corneal cell types (epithelial, stromal, and endothelial) together with neural cells. The neural cells were derived from hybrid neuroblastoma cells and the other cells used from immortalized human corneal cell lines. To check the feasibility and characterize the constructs, cytotoxicity, cell proliferation, histology, and protein expression studies were performed. Results: Optimized culture condition permitted synchronized viability across the cell types within the construct. The construct showed a typical appearance for different cellular layers, including healthy appearing, phenotypically differentiated neurons. The expected protein expression profiles for specific cell types within the construct were confirmed with western blotting. Conclusions: An in vitro corneal construct was successfully developed with maintenance of individual cell phenotypes with anatomically correct cellular loci. The construct may be useful in evaluation of specific corneal disorders and in developing different corneal disease models. Additionally, the construct can be used in evaluating drug targeting and/or penetration to individual corneal layers, testing novel therapeutics for corneal diseases, and potentially reducing the necessity for animals in corneal research at the early stages of investigation. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
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Alphaviruses are serious zoonotic threats responsible for significant morbidity, causing arthritis or encephalitis. So far, no licensed drugs or vaccines are available to combat alphaviral infections. About 300,000 chikungunya virus (CHIKV) infections have been reported in 2023, with more than 300 deaths, including reports of a few cases in the USA as well. The discovery and development of small-molecule drugs have been revolutionized over the last decade. Here, we employed a cell-based screening approach using a series of in-house small-molecule libraries to test for their ability to inhibit CHIKV replication. DCR 137, a quinazoline derivative, was found to be the most potent inhibitor of CHIKV replication in our screening assay. Both, the cytopathic effect, and immunofluorescence of infected cells were reduced in a dose-dependent manner with DCR 137 post-treatment. Most importantly, DCR 137 was more protective than the traditional ribavirin drug and reduced CHIKV plaque-forming units by several log units. CHIKV-E2 protein levels were also reduced in a dose-dependent manner. Further, DCR 137 was probed for its antiviral activity against another alphavirus, the Ross River virus, which revealed effective inhibition of viral replication. These results led to the identification of a potential quinazoline candidate for future optimization that might act as a pan-alphavirus inhibitor.
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Febre de Chikungunya , Vírus Chikungunya , Humanos , Ross River virus , Linhagem Celular , Antivirais/farmacologia , Vírus Chikungunya/fisiologia , Quinazolinas/farmacologia , Replicação ViralRESUMO
Heat shock proteins (HSPs) play a critical role in many intracellular processes, including apoptosis and delivery of other proteins to intracellular compartments. Small HSPs have been shown previously to participate in many cellular functions, including IL-8 induction. Human adenovirus infection activates intracellular signaling, involving particularly the c-Src and mitogen-activated protein kinases [Natarajan, K., et al. (2003) J. Immunol. 170, 6234-6243]. HSP27 and MK2 are also phosphorylated, and c-Src, and its downstream targets, p38, ERK1/2, and c-Jun-terminal kinase (JNK), differentially mediate IL-8 and MCP-1 expression. Specifically, activation and translocation of transcription factor NFκB-p65 occurs in a p38-dependent fashion [Rajaiya, J., et al. (2009) Mol. Vision 15, 2879-2889]. Herein, we report a novel role for HSP27 in an association of p38 with NFκB-p65. Immunoprecipitation assays of virus-infected but not mock-infected cells revealed a signaling complex including p38 and NFκB-p65. Transfection with HSP27 short interfering RNA (siRNA) but not scrambled RNA disrupted this association and reduced the level of IL-8 expression. Transfection with HSP27 siRNA also reduced the level of nuclear localization of NFκB-p65 and p38. By use of tagged p38 mutants, we found that amino acids 279-347 of p38 are necessary for the association of p38 with NFκB-p65. These studies strongly suggest that HSP27, p38, and NFκB-p65 form a signalosome in virus-infected cells and influence downstream expression of pro-inflammatory mediators.
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Infecções por Adenovirus Humanos/metabolismo , Adenovírus Humanos/fisiologia , Proteínas de Choque Térmico HSP27/fisiologia , Infecções por Adenovirus Humanos/virologia , Células Cultivadas , Quimiocinas/biossíntese , Humanos , Queratinócitos/virologia , Subunidade p50 de NF-kappa B/metabolismo , Transporte Proteico , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Human adenovirus (HAdV) infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9) signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9(-/-) mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD). These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins.
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Infecções por Adenoviridae/imunologia , Proteínas do Capsídeo/imunologia , Capsídeo/imunologia , Ceratite/virologia , Oligopeptídeos/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Infecções por Adenoviridae/genética , Animais , Separação Celular , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CCL2/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Expressão Gênica , Genes Virais , Humanos , Imuno-Histoquímica , Ceratite/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologiaRESUMO
Objective: To obtain complete DNA sequences of adenoviral (AdV) D8 genome from patients with conjunctivitis and determine the relation of sequence variation to clinical outcomes. Design: This study is a post hoc analysis of banked conjunctival swab samples from the BAYnovation Study, a previously conducted, randomized controlled clinical trial for AdV conjunctivitis. Participants: Ninety-six patients with AdV D8-positive conjunctivitis who received placebo treatment in the BAYnovation Study were included in the study. Methods: DNA from conjunctival swabs was purified and subjected to whole-genome viral DNA sequencing. Adenovirus D8 variants were identified and correlated with clinical outcomes, including 2 machine learning methods. Main Outcome Measures: Viral DNA sequence and development of subepithelial infiltrates (SEIs) were the main outcome measures. Results: From initial sequencing of 80 AdV D8-positive samples, full adenoviral genome reconstructions were obtained for 71. A total of 630 single-nucleotide variants were identified, including 156 missense mutations. Sequence clustering revealed 3 previously unappreciated viral clades within the AdV D8 type. The likelihood of SEI development differed significantly between clades, ranging from 83% for Clade 1 to 46% for Clade 3. Genome-wide analysis of viral single-nucleotide polymorphisms failed to identify single-gene determinants of outcome. Two machine learning models were independently trained to predict clinical outcome using polymorphic sequences. Both machine learning models correctly predicted development of SEI outcomes in a newly sequenced validation set of 16 cases (P = 1.5 × 10-5). Prediction was dependent on ensemble groups of polymorphisms across multiple genes. Conclusions: Adenovirus D8 has ≥ 3 prevalent molecular substrains, which differ in propensity to result in SEIs. Development of SEIs can be accurately predicted from knowledge of full viral sequence. These results suggest that development of SEIs in AdV D8 conjunctivitis is largely attributable to pathologic viral sequence variants within the D8 type and establishes machine learning paradigms as a powerful technique for understanding viral pathogenicity.
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Development of an artificial cornea can potentially fulfil the demand of donor corneas for transplantation as the number of donors is far less than needed to treat corneal blindness. Collagen-based artificial corneas stand out as a regenerative option, having promising clinical outcomes. Collagen crosslinked with chemical crosslinkers which modify the parent functional groups of collagen. However, crosslinkers are usually cytotoxic, so crosslinkers need to be removed from implants completely before application in humans. In addition, crosslinked products are mechanically weak and susceptible to enzymatic degradation. We developed a crosslinker free supramolecular gelation strategy using pyrene conjugated dipeptide amphiphile (PyKC) consisting of lysine and cysteine; in which collagen molecules are intertwined inside the PyKC network without any functional group modification of the collagen. The newly developed collagen implants (Coll-PyKC) are optically transparent and can effectively block UV light, are mechanically and enzymatically stable, and can be sutured. The Coll-PyKC implants support the growth and function of all corneal cells, trigger anti-inflammatory differentiation while suppressing the pro-inflammatory differentiation of human monocytes. Coll-PyKC implants can restrict human adenovirus propagation. Therefore, this crosslinker-free strategy can be used for the repair, healing, and regeneration of the cornea, and potentially other damaged organs of the body.
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Colágeno , Córnea , Colágeno/metabolismo , Córnea/metabolismo , Humanos , Próteses e Implantes , Regeneração , Raios UltravioletaRESUMO
Notable among the many communicable agents known to infect the human cornea is the human adenovirus, with less than ten adenoviruses having corneal tropism out of more than 100 known types. The syndrome of epidemic keratoconjunctivitis (EKC), caused principally by human adenovirus, presents acutely with epithelial keratitis, and later with stromal keratitis that can be chronic and recurrent. In this review, we discuss the current state of knowledge regarding the molecular biology of adenovirus infection of corneal stromal cells, among which the fibroblast-like keratocyte is the most predominant, in order to elucidate basic pathophysiologic mechanisms of stromal keratitis in the human patient with EKC.
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Adenovírus Humanos/fisiologia , Córnea/virologia , Ceratite/etiologia , Adenovírus Humanos/classificação , Animais , Córnea/citologia , Córnea/embriologia , Interações entre Hospedeiro e Microrganismos , Humanos , Interleucina-8/genética , Ceratoconjuntivite/etiologia , Organogênese , Células Estromais/virologiaRESUMO
Human adenoviruses cause disease at multiple mucosal sites, including the respiratory, gastrointestinal, and genitourinary tracts, and are common agents of conjunctivitis. One site of infection that has received sparse attention is the cornea, a transparent tissue and the window of the eye. While most adenovirus infections are self-limited, corneal inflammation (keratitis) due to adenovirus can persist or recur for months to years after infection, leading to reduced vision, discomfort, and light sensitivity. Topical corticosteroids effectively suppress late adenovirus keratitis but are associated with vision-threatening side effects. In this short review, we summarize current knowledge on infection of the cornea by adenoviruses, including corneal epithelial cell receptors and determinants of corneal tropism. We briefly discuss mechanisms of stromal keratitis due to adenovirus infection, and review an emerging therapy to mitigate adenovirus corneal infections based on evolving knowledge of corneal epithelial receptor usage.
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Infecções por Adenoviridae/virologia , Adenovírus Humanos/fisiologia , Córnea/virologia , Doenças da Córnea/virologia , Adenovírus Humanos/classificação , Adenovírus Humanos/genética , Adenovírus Humanos/isolamento & purificação , Animais , HumanosRESUMO
Recombination in human adenoviruses (HAdV) may confer virulence upon an otherwise nonvirulent strain. The genome sequence of species D HAdV type 22 (HAdV-D22) revealed evidence for recombination with HAdV-D19 and HAdV-D37 within the capsid penton base gene. Bootscan analysis demonstrated that recombination sites within the penton base gene frame the coding sequences for the two external hypervariable loops in the protein. A similar pattern of recombination was evident within other HAdV-D types but not other HAdV species. Further study of recombination among HAdVs is needed to better predict possible recombination events among wild-type viruses and adenoviral gene therapy vectors.
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Adenovírus Humanos/genética , Proteínas do Capsídeo/genética , DNA Viral/genética , Recombinação Genética , Sequência de Bases , Análise por Conglomerados , Evolução Molecular , Humanos , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
Known to occur in widespread outbreaks, epidemic keratoconjunctivitis (EKC) is a severe ocular surface infection with a strong historical association with human adenovirus (HAdV). While the conjunctival manifestations can vary from mild follicular conjunctivitis to hyper-acute, exudative conjunctivitis with formation of conjunctival membranes, EKC is distinct as the only form of adenovirus conjunctivitis in which the cornea is also involved, likely due to the specific corneal epithelial tropism of its causative viral agents. The initial development of a punctate or geographic epithelial keratitis may herald the later formation of stromal keratitis, and manifest as subepithelial infiltrates which often persist or recur for months to years after the acute infection has resolved. The chronic keratitis in EKC is associated with foreign body sensation, photophobia, glare, and reduced vision. However, over a century since the first clinical descriptions of EKC, and over 60 years since the first causative agent, human adenovirus type 8, was identified, our understanding of this disorder remains limited. This is underscored by a current lack of effective diagnostic tools and treatments. In part, stasis in our knowledge base has been encouraged by the continued acceptance, and indeed propagation of, inaccurate paradigms pertaining to disease etiology and pathogenesis, particularly with regard to mechanisms of innate and adaptive immunity within the cornea. Owing to its often persistent and medically refractory visual sequelae, reconsideration of key aspects of EKC disease biology is warranted to identify new treatment targets to curb its worldwide socioeconomic burden.
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Infecções por Adenovirus Humanos/epidemiologia , Adenovírus Humanos , Túnica Conjuntiva/virologia , Córnea/virologia , Infecções Oculares Virais/epidemiologia , Ceratoconjuntivite/epidemiologia , Infecções por Adenovirus Humanos/virologia , Epidemias , Infecções Oculares Virais/virologia , Saúde Global , Humanos , Ceratoconjuntivite/virologiaRESUMO
Purpose: Ocular infection by human adenovirus species D type 37 (HAdV-D37) causes epidemic keratoconjunctivitis, a severe, hyperacute condition. The corneal component of epidemic keratoconjunctivitis begins upon infection of corneal epithelium, and the mechanism of viral entry dictates subsequent proinflammatory gene expression. Therefore, it is important to understand the specific pathways of adenoviral entry in these cells. Methods: Transmission electron microscopy of primary and tert-immortalized human corneal epithelial cells infected with HAdV-D37 was performed to identify the means of viral entry. Confocal microscopy was used to determine intracellular trafficking. The results of targeted small interfering RNA and specific chemical inhibitors were analyzed by quantitative PCR, and Western blot. Results: By transmission electron microscopy, HAdV-D37 was seen to enter by both clathrin-coated pits and macropinocytosis; however, entry was both pH and dynamin 2 independent. Small interfering RNA against clathrin, AP2A1, and lysosome-associated membrane protein 1, but not early endosome antigen 1, decreased early viral gene expression. Ethyl-isopropyl amiloride, which blocks micropinocytosis, did not affect HAdV-D37 entry, but IPA, an inhibitor of p21-activated kinase, and important to actin polymerization, decreased viral entry in a dose-dependent manner. Conclusions: HAdV-D37 enters human corneal epithelial cells by a noncanonical clathrin-mediated pathway involving lysosome-associated membrane protein 1 and PAK1, independent of pH, dynamin, and early endosome antigen 1. We showed earlier that HAdV-D37 enters human keratocytes through caveolae. Therefore, epidemic keratoconjunctivitis-associated viruses enter different corneal cell types via disparate pathways, which could account for a relative paucity of proinflammatory gene expression upon infection of corneal epithelial cells compared with keratocytes, as seen in prior studies.
Assuntos
Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos , Epitélio Corneano/virologia , Ceratoconjuntivite Infecciosa/virologia , Internalização do Vírus , Adenovírus Humanos/fisiologia , Animais , Linhagem Celular , Vesículas Revestidas por Clatrina/virologia , Dinamina II/metabolismo , Epitélio Corneano/ultraestrutura , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Lisossomal/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Pinocitose , Reação em Cadeia da Polimerase em Tempo Real , Quinases Ativadas por p21/metabolismoRESUMO
PURPOSE: Corneal inflammation associated with ocular adenoviral infection is caused by leukocytic infiltration of the subepithelial stroma in response to expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by infected corneal cells. We have shown that these two chemokines are activated by the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38 for IL-8, and Jun-terminal kinase (JNK) for MCP-1. It is also well established that transcription of each of these chemokines is tightly controlled by the nuclear factor kappa B (NFkappaB) transcription factor family. Therefore, we sought to better understand the differential regulation of chemokine expression by NFkappaB in adenoviral infection of the cornea. METHODS: Primary keratocytes derived from human donor corneas were treated with signaling inhibitors and small interfering RNA specific to MAPKs, and infected with adenovirus for different time periods before analysis. Activation of specific NFkappaB subunits was analyzed by western blot, confocal microscopy, electromobility shift assay, and chromatin immunoprecipitation, and chemokine expression was quantified by enzyme-linked immunosorbent assay. RESULTS: Upon adenoviral infection, NFkappaB p65, p50, and cREL subunits translocate to the nucleus. This translocation is blocked by inhibitors of specific MAPK signaling pathways. Confocal microscopy showed that inhibitors of the p38, JNK, and ERK pathways differentially inhibited NFkappaB nuclear translocation, while PP2, an inhibitor of Src family kinases, completely inhibited NFkappaB nuclear translocation. Western blot analysis revealed that activation of specific NFkappaB subunits was time dependent following infection. Chromatin immunoprecipitation experiments indicated that binding of NFkappaB p65 and p50 subunits to the IL-8 promoter upon viral infection was differentially reduced by chemical inhibitors of MAPKs. Electromobility shift assay and luciferase assay analysis revealed that transactivation of IL-8 occurred with binding by the NFkappaB p65 homodimer or NFkappaB p65/p50 heterodimer as early as 1 h post infection, whereas MCP-1 expression was dependent upon the NFkappaB cREL but not the p65 subunit, and occurred 4 h after IL-8 induction. Finally, knockdown of NFkappaB p65 by short interfering RNA abrogated IL-8 but not MCP-1 expression after adenoviral infection. CONCLUSION: The kinetics of NFkappaB subunit activation are partly responsible for the observed pattern of acute inflammation in the adenoviral-infected cornea. MAPKs differentially regulate chemokine expression in adenoviral keratitis by differential and time-dependent activation of specific NFkappaB subunits.
Assuntos
Adenoviridae/fisiologia , Quimiocina CCL2/metabolismo , Interleucina-8/metabolismo , Ceratite/metabolismo , Ceratite/virologia , NF-kappa B/metabolismo , Subunidades Proteicas/metabolismo , Infecções por Adenoviridae/complicações , Infecções por Adenoviridae/enzimologia , Núcleo Celular/metabolismo , Quimiocina CCL2/genética , Imunoprecipitação da Cromatina , Ativação Enzimática , Humanos , Quinase I-kappa B/metabolismo , Interleucina-8/genética , Ceratite/complicações , Ceratite/enzimologia , Cinética , NF-kappa B/antagonistas & inibidores , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transporte Proteico , Fatores de Tempo , Fator de Transcrição RelA/metabolismoRESUMO
Bright/ARID3a/Dril1, a member of the ARID family of transcription factors, is expressed in a highly regulated fashion in B lymphocytes, where it enhances immunoglobulin transcription three- to sixfold. Recent publications from our lab indicated that functional, but not kinase-inactive, Bruton's tyrosine kinase (Btk) is critical for Bright activity in an in vitro model system, yet Bright itself is not appreciably tyrosine phosphorylated. These data suggested that a third protein, and Btk substrate, must contribute to Bright-enhanced immunoglobulin transcription. The ubiquitously expressed transcription factor TFII-I was identified as a substrate for Btk several years ago. In this work, we show that TFII-I directly interacts with human Bright through amino acids in Bright's protein interaction domain and that specific tyrosine residues of TFII-I are essential for Bright-induced activity of an immunoglobulin reporter gene. Moreover, inhibition of TFII-I function in a B-cell line resulted in decreased heavy-chain transcript levels. These data suggest that Bright functions as a three-component protein complex in the immunoglobulin locus and tie together previous data indicating important roles for Btk and TFII-I in B lymphocytes.