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1.
ACS Chem Biol ; 15(10): 2683-2691, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-32845119

RESUMO

Coxsackievirus A24 variant (CVA24v) and human adenovirus 37 (HAdV-37) are leading causative agents of the severe and highly contagious ocular infections acute hemorrhagic conjunctivitis and epidemic keratoconjunctivitis, respectively. Currently, neither vaccines nor antiviral agents are available for treating these diseases, which affect millions of individuals worldwide. CVA24v and HAdV-37 utilize sialic acid as attachment receptors facilitating entry into host cells. Previously, we and others have shown that derivatives based on sialic acid are effective in preventing HAdV-37 binding and infection of cells. Here, we designed and synthesized novel pentavalent sialic acid conjugates and studied their inhibitory effect against CVA24v and HAdV-37 binding and infection of human corneal epithelial cells. The pentavalent conjugates are the first reported inhibitors of CVA24v infection and proved efficient in blocking HAdV-37 binding. Taken together, the pentavalent conjugates presented here form a basis for the development of general inhibitors of these highly contagious ocular pathogens.

2.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376620

RESUMO

Virus entry into host cells is a complex process that is largely regulated by access to specific cellular receptors. Human adenoviruses (HAdVs) and many other viruses use cell adhesion molecules such as the coxsackievirus and adenovirus receptor (CAR) for attachment to and entry into target cells. These molecules are rarely expressed on the apical side of polarized epithelial cells, which raises the question of how adenoviruses-and other viruses that engage cell adhesion molecules-enter polarized cells from the apical side to initiate infection. We have previously shown that species C HAdVs utilize lactoferrin-a common innate immune component secreted to respiratory mucosa-for infection via unknown mechanisms. Using a series of biochemical, cellular, and molecular biology approaches, we mapped this effect to the proteolytically cleavable, positively charged, N-terminal 49 residues of human lactoferrin (hLF) known as human lactoferricin (hLfcin). Lactoferricin (Lfcin) binds to the hexon protein on the viral capsid and anchors the virus to an unknown receptor structure of target cells, resulting in infection. These findings suggest that HAdVs use distinct cell entry mechanisms at different stages of infection. To initiate infection, entry is likely to occur at the apical side of polarized epithelial cells, largely by means of hLF and hLfcin bridging HAdV capsids via hexons to as-yet-unknown receptors; when infection is established, progeny virions released from the basolateral side enter neighboring cells by means of hLF/hLfcin and CAR in parallel.IMPORTANCE Many viruses enter target cells using cell adhesion molecules as receptors. Paradoxically, these molecules are abundant on the lateral and basolateral side of intact, polarized, epithelial target cells, but absent on the apical side that must be penetrated by incoming viruses to initiate infection. Our study provides a model whereby viruses use different mechanisms to infect polarized epithelial cells depending on which side of the cell-apical or lateral/basolateral-is attacked. This study may also be useful to understand the biology of other viruses that use cell adhesion molecules as receptors.


Assuntos
Infecções por Adenovirus Humanos/metabolismo , Adenovírus Humanos/metabolismo , Proteínas do Capsídeo/metabolismo , Células Epiteliais/metabolismo , Lactoferrina/metabolismo , Mucosa Respiratória/metabolismo , Células A549 , Infecções por Adenovirus Humanos/genética , Adenovírus Humanos/genética , Proteínas do Capsídeo/genética , Células Epiteliais/virologia , Humanos , Lactoferrina/genética , Mucosa Respiratória/virologia
3.
PLoS One ; 14(6): e0217359, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31163044

RESUMO

Tick-borne encephalitis infections have increased the last 30 years. The mortality associated to this viral infection is 0.5 to 30% with a risk of permanent neurological sequelae, however, no therapeutic is currently available. The first steps of virus-cell interaction, such as attachment and entry, are of importance to understand pathogenesis and tropism. Several molecules have been shown to interact with tick-borne encephalitis virus (TBEV) at the plasma membrane surface, yet, no studies have proven that these are specific entry receptors. In this study, we set out to characterize the cellular attachment receptor(s) for TBEV using the naturally attenuated member of the TBEV complex, Langat virus (LGTV), as a model. Inhibiting or cleaving different molecules from the surface of A549 cells, combined with inhibition assays using peptide extracts from high LGTV binding cells, revealed that LGTV attachment to host cells is dependent on plasma membrane proteins, but not on glycans or glycolipids, and suggested that LGTV might use different cellular attachment factors on different cell types. Based on this, we developed a transcriptomic approach to generate a list of candidate attachment and entry receptors. Our findings shed light on the first step of the flavivirus life-cycle and provide candidate receptors that might serve as a starting point for future functional studies to identify the specific attachment and/or entry receptor for LGTV and TBEV.


Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/metabolismo , Encefalite Transmitida por Carrapatos/metabolismo , Proteínas de Membrana/metabolismo , Ligação Viral , Internalização do Vírus , Células A549 , Humanos
4.
Viruses ; 11(5)2019 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-31035532

RESUMO

Human adenoviruses (HAdV) are the most common cause of ocular infections. Species B human adenovirus type 3 (HAdV-B3) causes pharyngoconjunctival fever (PCF), whereas HAdV-D8, -D37, and -D64 cause epidemic keratoconjunctivitis (EKC). Recently, HAdV-D53, -D54, and -D56 emerged as new EKC-causing agents. HAdV-E4 is associated with both PCF and EKC. We have previously demonstrated that HAdV-D37 uses sialic acid (SA)-containing glycans as cellular receptors on human corneal epithelial (HCE) cells, and the virus interaction with SA is mediated by the knob domain of the viral fiber protein. Here, by means of cell-based assays and using neuraminidase (a SA-cleaving enzyme), we investigated whether ocular HAdVs other than HAdV-D37 also use SA-containing glycans as receptors on HCE cells. We found that HAdV-E4 and -D56 infect HCE cells independent of SAs, whereas HAdV-D53 and -D64 use SAs as cellular receptors. HAdV-D8 and -D54 fiber knobs also bound to cell-surface SAs. Surprisingly, HCE cells were found resistant to HAdV-B3 infection. We also demonstrated that the SA-based molecule i.e., ME0462, designed to bind to SA-binding sites on the HAdV-D37 fiber knob, efficiently prevents binding and infection of several EKC-causing HAdVs. Surface plasmon resonance analysis confirmed a direct interaction between ME0462 and fiber knobs. Altogether, we demonstrate that SA-containing glycans serve as receptors for multiple EKC-causing HAdVs, and, that SA-based compound function as a broad-spectrum antiviral against known and emerging EKC-causing HAdVs.


Assuntos
Infecções por Adenovirus Humanos/metabolismo , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/fisiologia , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Receptores Virais/metabolismo , Tropismo Viral , Células A549 , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Humanos , Ceratoconjuntivite/metabolismo , Ceratoconjuntivite/virologia , Análise de Sequência de DNA
5.
Viruses ; 11(3)2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30870979

RESUMO

Epidemic keratoconjunctivitis (EKC) is a severe ocular disease and can lead to visual impairment. Human adenovirus type-37 (HAdV-D37) is one of the major causative agents of EKC and uses sialic acid (SA)-containing glycans as cellular receptors. Currently, there are no approved antivirals available for the treatment of EKC. Recently, we have reported that sulfated glycosaminoglycans (GAGs) bind to HAdV-D37 via the fiber knob (FK) domain of the viral fiber protein and function as decoy receptors. Based on this finding, we speculated that GAG-mimetics may act as artificial decoy receptors and inhibit HAdV-D37 infection. Repurposing of approved drugs to identify new antivirals has drawn great attention in recent years. Here, we report the antiviral effect of suramin, a WHO-approved drug and a widely known GAG-mimetic, against HAdV-D37. Commercially available suramin analogs also show antiviral effects against HAdV-D37. We demonstrate that suramin exerts its antiviral activity by inhibiting the attachment of HAdV-D37 to cells. We also reveal that the antiviral effect of suramin is HAdV species-specific. Collectively, in this proof of concept study, we demonstrate for the first time that virus binding to a decoy receptor constitutes a novel and an unexplored target for antiviral drug development.


Assuntos
Adenovírus Humanos/efeitos dos fármacos , Ceratoconjuntivite/virologia , Receptores Virais/metabolismo , Suramina/farmacologia , Ligação Viral/efeitos dos fármacos , Células A549 , Infecções por Adenovirus Humanos/tratamento farmacológico , Infecções por Adenovirus Humanos/virologia , Sítios de Ligação , DNA Viral , Reposicionamento de Medicamentos , Genoma Viral , Humanos , Ceratoconjuntivite/tratamento farmacológico , Filogenia , Estudo de Prova de Conceito , Análise de Sequência de DNA
6.
Viruses ; 11(3)2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30871026

RESUMO

Glycans on plasma membranes and in secretions play important roles in infection by many viruses. Species D human adenovirus type 37 (HAdV-D37) is a major cause of epidemic keratoconjunctivitis (EKC) and infects target cells by interacting with sialic acid (SA)-containing glycans via the fiber knob domain of the viral fiber protein. HAdV-D37 also interacts with sulfated glycosaminoglycans (GAGs), but the outcome of this interaction remains unknown. Here, we investigated the molecular requirements of HAdV-D37 fiber knob:GAG interactions using a GAG microarray and demonstrated that fiber knob interacts with a broad range of sulfated GAGs. These interactions were corroborated in cell-based assays and by surface plasmon resonance analysis. Removal of heparan sulfate (HS) and sulfate groups from human corneal epithelial (HCE) cells by heparinase III and sodium chlorate treatments, respectively, reduced HAdV-D37 binding to cells. Remarkably, removal of HS by heparinase III enhanced the virus infection. Our results suggest that interaction of HAdV-D37 with sulfated GAGs in secretions and on plasma membranes prevents/delays the virus binding to SA-containing receptors and inhibits subsequent infection. We also found abundant HS in the basement membrane of the human corneal epithelium, which may act as a barrier to sub-epithelial infection. Collectively, our findings provide novel insights into the role of GAGs as viral decoy receptors and highlight the therapeutic potential of GAGs and/or GAG-mimetics in HAdV-D37 infection.


Assuntos
Adenovírus Humanos/química , Glicosaminoglicanos/química , Heparitina Sulfato/química , Receptores Virais/química , Células A549 , Adenovírus Humanos/genética , DNA Viral/genética , Epitélio Anterior/química , Epitélio Anterior/virologia , Genoma Viral , Glicosaminoglicanos/genética , Humanos , Análise em Microsséries , Filogenia , Receptores Virais/genética , Proteínas Virais/genética , Tropismo Viral , Ligação Viral
7.
PLoS One ; 13(12): e0209038, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30550551

RESUMO

Human adenoviruses (HAdVs) are common pathogens that can cause respiratory, gastrointestinal, urogenital, and ocular infections. They are divided into seven species containing 85 genotypes. Straightforward typing systems might help epidemiological investigations. As homologous recombination frequently shapes the evolution of HAdVs, information on a single gene is seldom sufficient to allow accurate and precise typing, and complete genome-based methods are recommended. Even so, complete genome analyses are not always easy to perform for practical reasons, and in such cases a multigene system can provide considerably more information about the strain under investigation than single-gene-based methods. Here we present a rapid, generic, multigene typing system for HAdVs based on three main deterministic regions of these viruses. Three PCR systems were used to amplify the genes encoding the DNA polymerase, the penton base hypervariable Arg-Gly-Asp-containing loop, and the hexon loop 1 (hypervariable region 1-6). Using this system, we typed 281 clinical isolates, detected members of six out of seven HAdV species (Human mastadenovirus A-F), and could also detect not only divergent strains of established types but also a new recombinant strain with a previously unpublished combination of adenovirus genomes. This strain was accepted by the Human Adenovirus Working Group as a novel genotype: HAdV-86. Seven strains that could not be typed with sufficient accuracy were also investigated using a PCR based on part of the fiber gene. By analysis of corresponding sequences of the 86 known HAdV genotypes, we determined that the proposed typing system should be able to distinguish all non-recombinant types, and with additional fiber information, all known HAdV genotypes.


Assuntos
Adenovírus Humanos/genética , Genes Virais/genética , Genótipo , Família Multigênica/genética , Adenovírus Humanos/classificação , Genômica , Humanos , Filogenia , Reação em Cadeia da Polimerase , Suécia
8.
Sci Rep ; 8(1): 10019, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29968781

RESUMO

The enteric species F human adenovirus types 40 and 41 (HAdV-40 and -41) are the third most common cause of infantile gastroenteritis in the world. Knowledge about HAdV-40 and -41 cellular infection is assumed to be fundamentally different from that of other HAdVs since HAdV-40 and -41 penton bases lack the αV-integrin-interacting RGD motif. This motif is used by other HAdVs mainly for internalization and endosomal escape. We hypothesised that the penton bases of HAdV-40 and -41 interact with integrins independently of the RGD motif. HAdV-41 transduction of a library of rodent cells expressing specific human integrin subunits pointed to the use of laminin-binding α2-, α3- and α6-containing integrins as well as other integrins as candidate co-receptors. Specific laminins prevented internalisation and infection, and recombinant, soluble HAdV-41 penton base proteins prevented infection of human intestinal HT-29 cells. Surface plasmon resonance analysis demonstrated that HAdV-40 and -41 penton base proteins bind to α6-containing integrins with an affinity similar to that of previously characterised penton base:integrin interactions. With these results, we propose that laminin-binding integrins are co-receptors for HAdV-40 and -41.


Assuntos
Adenovírus Humanos/metabolismo , Integrina alfa6/metabolismo , Integrina alfa6beta4/metabolismo , Laminina/metabolismo , Receptores Virais/metabolismo , Ligação Viral , Animais , Células CHO , Linhagem Celular , Cricetulus , Células HT29 , Humanos , Ressonância de Plasmônio de Superfície
9.
J Mol Biol ; 430(13): 1863-1882, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29746851

RESUMO

Adenoviruses as most viruses rely on glycan and protein interactions to attach to and enter susceptible host cells. The Adenoviridae family comprises more than 80 human types and they differ in their attachment factor and receptor usage, which likely contributes to the diverse tropism of the different types. In the past years, methods to systematically identify glycan and protein interactions have advanced. In particular sensitivity, speed and coverage of mass spectrometric analyses allow for high-throughput identification of glycans and peptides separated by liquid chromatography. Also, developments in glycan microarray technologies have led to targeted, high-throughput screening and identification of glycan-based receptors. The mapping of cell surface interactions of the diverse adenovirus types has implications for cell, tissue, and species tropism as well as drug development. Here we review known adenovirus interactions with glycan- and protein-based receptors, as well as glycomics and proteomics strategies to identify yet elusive virus receptors and attachment factors. We finally discuss challenges, bottlenecks, and future research directions in the field of non-enveloped virus entry into host cells.


Assuntos
Infecções por Adenoviridae/metabolismo , Adenoviridae/fisiologia , Glicômica/métodos , Proteômica/métodos , Interações entre Hospedeiro e Microrganismos , Humanos , Análise em Microsséries , Polissacarídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Tropismo Viral
10.
Proc Natl Acad Sci U S A ; 115(18): E4264-E4273, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29674446

RESUMO

Human adenovirus 52 (HAdV-52) is one of only three known HAdVs equipped with both a long and a short fiber protein. While the long fiber binds to the coxsackie and adenovirus receptor, the function of the short fiber in the virus life cycle is poorly understood. Here, we show, by glycan microarray analysis and cellular studies, that the short fiber knob (SFK) of HAdV-52 recognizes long chains of α-2,8-linked polysialic acid (polySia), a large posttranslational modification of selected carrier proteins, and that HAdV-52 can use polySia as a receptor on target cells. X-ray crystallography, NMR, molecular dynamics simulation, and structure-guided mutagenesis of the SFK reveal that the nonreducing, terminal sialic acid of polySia engages the protein with direct contacts, and that specificity for polySia is achieved through subtle, transient electrostatic interactions with additional sialic acid residues. In this study, we present a previously unrecognized role for polySia as a cellular receptor for a human viral pathogen. Our detailed analysis of the determinants of specificity for this interaction has general implications for protein-carbohydrate interactions, particularly concerning highly charged glycan structures, and provides interesting dimensions on the biology and evolution of members of Human mastadenovirus G.


Assuntos
Adenovírus Humanos/química , Simulação de Dinâmica Molecular , Ácidos Siálicos/química , Adenovírus Humanos/metabolismo , Linhagem Celular Tumoral , Humanos , Ácidos Siálicos/metabolismo
11.
J Virol ; 92(7)2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29367250

RESUMO

Human adenovirus 41 (HAdV-41) causes acute gastroenteritis in young children. The main characteristics of HAdV-41 infection are diarrhea and vomiting. Nevertheless, the precise mechanism of HAdV-41-induced diarrhea is unknown, as a suitable small-animal model has not been described. In this study, we used the human midgut carcinoid cell line GOT1 to investigate the effect of HAdV-41 infection and the individual HAdV-41 capsid proteins on serotonin release by enterochromaffin cells and on enteric glia cell (EGC) activation. We first determined that HAdV-41 could infect the enterochromaffin cells. Immunofluorescence staining revealed that the cells expressed HAdV-41-specific coxsackievirus and adenovirus receptor (CAR); flow cytometry analysis supported these findings. HAdV-41 infection of the enterochromaffin cells induced serotonin secretion dose dependently. In contrast, control infection with HAdV-5 did not induce serotonin secretion in the cells. Confocal microscopy studies of enterochromaffin cells infected with HAdV-41 revealed decreased serotonin immunofluorescence compared to that in uninfected cells. Incubation of the enterochromaffin cells with purified HAdV-41 short fiber knob and hexon proteins increased the serotonin levels in the harvested cell supernatant significantly. HAdV-41 infection could also activate EGCs, as shown in the significantly altered expression of glia fibrillary acidic protein (GFAP) in EGCs incubated with HAdV-41. The EGCs were also activated by serotonin alone, as shown in the significantly increased GFAP staining intensity. Likewise, EGCs were activated by the cell supernatant of HAdV-41-infected enterochromaffin cells.IMPORTANCE The nonenveloped human adenovirus 41 causes diarrhea, vomiting, dehydration, and low-grade fever mainly in children under 2 years of age. Even though acute gastroenteritis is well described, how human adenovirus 41 causes diarrhea is unknown. In our study, we analyzed the effect of human adenovirus 41 infection on human enterochromaffin cells and found it stimulates serotonin secretion in the cells, which is involved in regulation of intestinal secretion and gut motility and can also activate enteric glia cells, which are found in close proximity to enterochromaffin cells in vivo This disruption of gut barrier homeostasis as maintained by these cells following human adenovirus 41 infection might be a mechanism in enteric adenovirus pathogenesis in humans and could indicate a possible serotonin-dependent cross talk between human adenovirus 41, enterochromaffin cells, and enteric glia cells.


Assuntos
Infecções por Adenoviridae/metabolismo , Adenoviridae/metabolismo , Células Enterocromafins/metabolismo , Neuroglia/metabolismo , Serotonina/metabolismo , Células A549 , Infecções por Adenoviridae/patologia , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Células Enterocromafins/patologia , Células Enterocromafins/virologia , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Neuroglia/patologia , Neuroglia/virologia
12.
Proc Natl Acad Sci U S A ; 115(2): 397-402, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29284752

RESUMO

Acute hemorrhagic conjunctivitis (AHC) is a painful, contagious eye disease, with millions of cases in the last decades. Coxsackievirus A24 (CV-A24) was not originally associated with human disease, but in 1970 a pathogenic "variant" (CV-A24v) emerged, which is now the main cause of AHC. Initially, this variant circulated only in Southeast Asia, but it later spread worldwide, accounting for numerous AHC outbreaks and two pandemics. While both CV-A24 variant and nonvariant strains still circulate in humans, only variant strains cause AHC for reasons that are yet unknown. Since receptors are important determinants of viral tropism, we set out to map the CV-A24 receptor repertoire and establish whether changes in receptor preference have led to the increased pathogenicity and rapid spread of CV-A24v. Here, we identify ICAM-1 as an essential receptor for both AHC-causing and non-AHC strains. We provide a high-resolution cryo-EM structure of a virus-ICAM-1 complex, which revealed critical ICAM-1-binding residues. These data could help identify a possible conserved mode of receptor engagement among ICAM-1-binding enteroviruses and rhinoviruses. Moreover, we identify a single capsid substitution that has been adopted by all pandemic CV-A24v strains and we reveal that this adaptation enhances the capacity of CV-A24v to bind sialic acid. Our data elucidate the CV-A24v receptor repertoire and point to a role of enhanced receptor engagement in the adaptation to the eye, possibly enabling pandemic spread.


Assuntos
Conjuntivite Hemorrágica Aguda/metabolismo , Enterovirus Humano C/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Receptores Virais/metabolismo , Sequência de Aminoácidos , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Conjuntivite Hemorrágica Aguda/epidemiologia , Conjuntivite Hemorrágica Aguda/virologia , Microscopia Crioeletrônica , Surtos de Doenças , Enterovirus Humano C/genética , Enterovirus Humano C/fisiologia , Humanos , Molécula 1 de Adesão Intercelular/química , Mutação , Ácido N-Acetilneuramínico/metabolismo , Pandemias , Filogenia , Ligação Proteica , Receptores Virais/química , Homologia de Sequência de Aminoácidos , Tropismo Viral/fisiologia
13.
J Gen Virol ; 99(1): 135-147, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29154744

RESUMO

The vectorization of rare human adenovirus (HAdV) types will widen our knowledge of this family and their interaction with cells, tissues and organs. In this study we focus on HAdV-56, a member of human Ad species D, and create ease-of-use cloning systems to generate recombinant HAdV-56 vectors carrying foreign genes. We present in vitro transduction profiles for HAdV-56 in direct comparison to the most commonly used HAdV-5-based vector. In vivo characterizations demonstrate that when it is delivered intravenously (i.v.) HAdV-56 mainly targets the spleen and, to a lesser extent, the lungs, whilst largely bypassing liver transduction in mice. HAdV-56 triggered robust inflammatory and cellular immune responses, with higher induction of IFNγ, TNFα, IL5, IL6, IP10, MCP1 and MIG1 compared to HAdV-5 following i.v. administration. We also investigated its potential as a vaccine vector candidate by performing prime immunizations in mice with HAdV-56 encoding luciferase (HAdV-56-Luc). Direct comparisons were made to HAdV-26, a highly potent human vaccine vector currently in phase II clinical trials. HAdV-56-Luc induced luciferase 'antigen'-specific IFNγ-producing cells and anti-HAdV-56 neutralizing antibodies in Balb/c mice, demonstrating a near identical profile to that of HAdV-26. Taken together, the data presented provides further insight into human Ad receptor/co-receptor usage, and the first report on HAdV-56 vectors and their potential for gene therapy and vaccine applications.


Assuntos
Adenovírus Humanos/imunologia , Expressão Gênica/efeitos dos fármacos , Terapia Genética/métodos , Vetores Genéticos/imunologia , Vacinação , Vacinas Virais/biossíntese , Adenovírus Humanos/genética , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Quimiocina CXCL9/genética , Quimiocina CXCL9/imunologia , Feminino , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Injeções Intravenosas , Interferon gama/genética , Interferon gama/imunologia , Interleucina-5/genética , Interleucina-5/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/efeitos dos fármacos , Baço/imunologia , Transgenes , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Vacinas Virais/administração & dosagem
14.
Virus Res ; 242: 79-84, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28923509

RESUMO

A novel human adenovirus was isolated from a pediatric case of acute respiratory disease in Panama City, Panama in 2011. The clinical isolate was initially identified as an intertypic recombinant based on hexon and fiber gene sequencing. Based on the analysis of its complete genome sequence, the novel complex recombinant Human mastadenovirus D (HAdV-D) strain was classified into a new HAdV type: HAdV-84, and it was designated Adenovirus D human/PAN/P309886/2011/84[P43H17F84]. HAdV-D types possess usually an ocular or gastrointestinal tropism, and respiratory association is scarcely reported. The virus has a novel fiber type, most closely related to, but still clearly distant from that of HAdV-36. The predicted fiber is hypothesised to bind sialic acid with lower affinity compared to HAdV-37. Bioinformatic analysis of the complete genomic sequence of HAdV-84 revealed multiple homologous recombination events and provided deeper insight into HAdV evolution.


Assuntos
Adenovírus Humanos/genética , Proteínas do Capsídeo/genética , Genoma Viral , Genótipo , Análise de Sequência de DNA , Infecções por Adenoviridae/virologia , Adenovírus Humanos/isolamento & purificação , Proteínas do Capsídeo/metabolismo , Humanos , Panamá , Filogenia , Ligação Proteica , Recombinação Genética , Homologia de Sequência , Ácidos Siálicos/metabolismo
15.
J Virol ; 91(5)2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-27974569

RESUMO

Epidemic keratoconjunctivitis (EKC) is a severe, contagious ocular disease that affects 20 to 40 million individuals worldwide every year. EKC is mainly caused by six types of human adenovirus (HAdV): HAdV-8, -19, -37, -53, -54, and -56. Of these, HAdV-8, -19, and -37 use sialic acid-containing glycans as cellular receptors. αVß3, αVß5, and a few additional integrins facilitate entry and endosomal release of other HAdVs. With the exception of a few biochemical analyses indicating that HAdV-37 can interact physically with αVß5, little is known about the integrins used by EKC-causing HAdVs. Here, we investigated the overall integrin expression on human corneal cells and found expression of α2, α3, α6, αV, ß1, and ß4 subunits in human corneal in situ epithelium and/or in a human corneal epithelial (HCE) cell line but no or less accessible expression of α4, α5, ß3, or ß5. We also identified the integrins used by HAdV-37 through a series of binding and infection competition experiments and different biochemical approaches. Together, our data suggest that HAdV-37 uses αVß1 and α3ß1 integrins for infection of human corneal epithelial cells. Furthermore, to confirm the relevance of these integrins in the HAdV-37 life cycle, we developed a corneal multilayer tissue system and found that HAdV-37 infection correlated well with the patterns of αV, α3, and ß1 integrin expression. These results provide further insight into the tropism and pathogenesis of EKC-causing HAdVs and may be of importance for future development of new antiviral drugs.IMPORTANCE Keratitis is a hallmark of EKC, which is caused by six HAdV types (HAdV-8, -19, -37, -53, -54, and -56). HAdV-37 and some other HAdV types interact with integrin αVß5 in order to enter nonocular human cells. In this study, we found that αVß5 is not expressed on human corneal epithelial cells, thus proposing other host factors mediate corneal infection. Here, we first characterized integrin expression patterns on corneal tissue and corneal cells. Among the integrins identified, competition binding and infection experiments and biochemical assays pointed out αVß1 and α3ß1 to be of importance for HAdV-37 infection of corneal tissue. In the absence of a good animal model for EKC-causing HAdVs, we also developed an in vitro system with multilayer HCE cells and confirmed the relevance of the suggested integrins during HAdV-37 infection.


Assuntos
Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/fisiologia , Integrina alfa3beta1/fisiologia , Receptores de Vitronectina/fisiologia , Células A549 , Córnea/patologia , Córnea/virologia , Humanos , Receptores Virais , Ligação Viral , Internalização do Vírus
16.
Org Biomol Chem ; 13(35): 9194-205, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26177934

RESUMO

Adenovirus type 37 (Ad37) is one of the principal agents responsible for epidemic keratoconjunctivitis (EKC), a severe ocular infection that remains without any available treatment. Recently, a trivalent sialic acid derivative (ME0322, Angew. Chem. Int. Ed., 2011, 50, 6519) was shown to function as a highly potent inhibitor of Ad37, efficiently preventing the attachment of the virion to the host cells and subsequent infection. Here, new trivalent sialic acid derivatives were designed, synthesized and their inhibitory properties against Ad37 infection of the human corneal epithelial cells were investigated. In comparison to ME0322, the best compound (17a) was found to be over three orders of magnitude more potent in a cell-attachment assay (IC50 = 1.4 nM) and about 140 times more potent in a cell-infection assay (IC50 = 2.9 nM). X-ray crystallographic analysis demonstrated a trivalent binding mode of all compounds to the Ad37 fiber knob. For the most potent compound ophthalmic toxicity in rabbits was investigated and it was concluded that repeated eye administration did not cause any adverse effects.


Assuntos
Adenoviridae/efeitos dos fármacos , Adenoviridae/fisiologia , Córnea/citologia , Células Epiteliais/virologia , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/farmacologia , Triazóis/química , Animais , Antivirais/síntese química , Antivirais/química , Antivirais/farmacologia , Química Click , Desenho de Fármacos , Células Epiteliais/efeitos dos fármacos , Humanos , Masculino , Modelos Moleculares , Conformação Molecular , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/síntese química , Coelhos
17.
PLoS Pathog ; 11(2): e1004657, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25674795

RESUMO

Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR) protein. Human adenovirus type 52 (HAdV-52) is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK) binds to CAR, and the knob domain of the short fiber (52SFK) binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy.


Assuntos
Adenovírus Humanos , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Glicoproteínas , Proteínas Virais , Tropismo Viral/fisiologia , Ligação Viral , Adenovírus Humanos/química , Adenovírus Humanos/fisiologia , Sequência de Bases , Linhagem Celular , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/química , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Cristalografia por Raios X , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Virais/química , Proteínas Virais/metabolismo
18.
PLoS Pathog ; 10(10): e1004401, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25329320

RESUMO

The picornaviruses coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac) for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 Å resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC.


Assuntos
Enterovirus Humano C/química , Picornaviridae/química , Receptores Virais/imunologia , Ácidos Siálicos/química , Sítios de Ligação , Linhagem Celular , Humanos , Receptores Virais/metabolismo
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