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1.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33384338

RESUMO

Human adenovirus species D (HAdV-D) types are currently being explored as vaccine vectors for coronavirus disease 2019 (COVID-19) and other severe infectious diseases. The efficacy of such vector-based vaccines depends on functional interactions with receptors on host cells. Adenoviruses of different species are assumed to enter host cells mainly by interactions between the knob domain of the protruding fiber capsid protein and cellular receptors. Using a cell-based receptor-screening assay, we identified CD46 as a receptor for HAdV-D56. The function of CD46 was validated in infection experiments using cells lacking and overexpressing CD46, and by competition infection experiments using soluble CD46. Remarkably, unlike HAdV-B types that engage CD46 through interactions with the knob domain of the fiber protein, HAdV-D types infect host cells through a direct interaction between CD46 and the hexon protein. Soluble hexon proteins (but not fiber knob) inhibited HAdV-D56 infection, and surface plasmon analyses demonstrated that CD46 binds to HAdV-D hexon (but not fiber knob) proteins. Cryoelectron microscopy analysis of the HAdV-D56 virion-CD46 complex confirmed the interaction and showed that CD46 binds to the central cavity of hexon trimers. Finally, soluble CD46 inhibited infection by 16 out of 17 investigated HAdV-D types, suggesting that CD46 is an important receptor for a large group of adenoviruses. In conclusion, this study identifies a noncanonical entry mechanism used by human adenoviruses, which adds to the knowledge of adenovirus biology and can also be useful for development of adenovirus-based vaccine vectors.


Assuntos
Adenovírus Humanos , Proteínas do Capsídeo , Regulação Viral da Expressão Gênica , Internalização do Vírus , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , /metabolismo , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Linhagem Celular , Humanos
2.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641484

RESUMO

Human adenoviruses (HAdV) are ubiquitous within the human population and comprise a significant burden of respiratory illnesses worldwide. Pediatric and immunocompromised individuals are at particular risk for developing severe disease; however, no approved antiviral therapies specific to HAdV exist. Ivermectin is an FDA-approved broad-spectrum antiparasitic drug that also exhibits antiviral properties against a diverse range of viruses. Its proposed function is inhibiting the classical protein nuclear import pathway mediated by importin-α (Imp-α) and -ß1 (Imp-ß1). Many viruses, including HAdV, rely on this host pathway for transport of viral proteins across the nuclear envelope. In this study, we show that ivermectin inhibits HAdV-C5 early gene transcription, early and late protein expression, genome replication, and production of infectious viral progeny. Similarly, ivermectin inhibits genome replication of HAdV-B3, a clinically important pathogen responsible for numerous recent outbreaks. Mechanistically, we show that ivermectin disrupts binding of the viral E1A protein to Imp-α without affecting the interaction between Imp-α and Imp-ß1. Our results further extend ivermectin's broad antiviral activity and provide a mechanistic underpinning for its mode of action as an inhibitor of cellular Imp-α/ß1-mediated nuclear import.IMPORTANCE Human adenoviruses (HAdVs) represent a ubiquitous and clinically important pathogen without an effective antiviral treatment. HAdV infections typically cause mild symptoms; however, individuals such as children, those with underlying conditions, and those with compromised immune systems can develop severe disseminated disease. Our results demonstrate that ivermectin, an FDA-approved antiparasitic agent, is effective at inhibiting replication of several HAdV types in vitro This is in agreement with the growing body of literature suggesting ivermectin has broad antiviral activity. This study expands our mechanistic knowledge of ivermectin by showing that ivermectin targets the ability of importin-α (Imp-α) to recognize nuclear localization sequences, without effecting the Imp-α/ß1 interaction. These data also exemplify the applicability of targeting host factors upon which viruses rely as a viable antiviral strategy.


Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Adenovírus Humanos/efeitos dos fármacos , Antiparasitários/farmacologia , Ivermectina/farmacologia , Replicação Viral/efeitos dos fármacos , alfa Carioferinas/genética , beta Carioferinas/genética , Células A549 , Transporte Ativo do Núcleo Celular/genética , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Adenovírus Humanos/patogenicidade , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Citosol/virologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Regulação da Expressão Gênica , Células HEK293 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Transdução de Sinais , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Proteínas Virais/metabolismo , alfa Carioferinas/antagonistas & inibidores , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismo
3.
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
4.
Proc Natl Acad Sci U S A ; 117(24): 13699-13707, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32467158

RESUMO

Adenovirus minor coat protein VI contains a membrane-disrupting peptide that is inactive when VI is bound to hexon trimers. Protein VI must be released during entry to ensure endosome escape. Hexon:VI stoichiometry has been uncertain, and only fragments of VI have been identified in the virion structure. Recent findings suggest an unexpected relationship between VI and the major core protein, VII. According to the high-resolution structure of the mature virion, VI and VII may compete for the same binding site in hexon; and noninfectious human adenovirus type 5 particles assembled in the absence of VII (Ad5-VII-) are deficient in proteolytic maturation of protein VI and endosome escape. Here we show that Ad5-VII- particles are trapped in the endosome because they fail to increase VI exposure during entry. This failure was not due to increased particle stability, because capsid disruption happened at lower thermal or mechanical stress in Ad5-VII- compared to wild-type (Ad5-wt) particles. Cryoelectron microscopy difference maps indicated that VII can occupy the same binding pocket as VI in all hexon monomers, strongly arguing for binding competition. In the Ad5-VII- map, density corresponding to the immature amino-terminal region of VI indicates that in the absence of VII the lytic peptide is trapped inside the hexon cavity, and clarifies the hexon:VI stoichiometry conundrum. We propose a model where dynamic competition between proteins VI and VII for hexon binding facilitates the complete maturation of VI, and is responsible for releasing the lytic protein from the hexon cavity during entry and stepwise uncoating.


Assuntos
Adenovírus Humanos/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Montagem de Vírus , Internalização do Vírus , Adenovírus Humanos/genética , Adenovírus Humanos/ultraestrutura , Microscopia Crioeletrônica , Humanos , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Ligação Proteica , Domínios Proteicos
5.
Virology ; 546: 67-78, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32452418

RESUMO

The E3 region of all simian and human types classified within species Human mastadenovirus B (HAdV-B) encodes two unique highly conserved ORFs of unknown function designated E3-CR1ß and E3-CR1γ. We generated a HAdV-3 mutant encoding small epitope tags at the N-termini of both E3-CR1ß and E3-CR1γ (HAdV-3 N-tag wt) and a double knock out (HAdV-3 N-tag DKO) mutant virus that does not express either protein. Our studies show that HAdV-3 E3-CR1ß and E3-CR1γ are type I transmembrane proteins that are produced predominantly at late times post infection, are glycosylated, co-localize at the plasma membrane of non-polarized epithelial cells, and interact with each other. At their extreme C-termini HAdV-B E3-CR1ß and E3-CR1γ possess a conserved di-leucine motif followed by a class II PDZ domain binding motif (PBM). HAdV-3 E3-CR1ß and E3-CR1γ are dispensable for virus growth, progeny release, spread, and plaque formation in A549 cells.


Assuntos
Proteínas E3 de Adenovirus/química , Proteínas E3 de Adenovirus/metabolismo , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/metabolismo , Membrana Celular/virologia , Proteínas E3 de Adenovirus/genética , Adenovírus Humanos/química , Adenovírus Humanos/genética , Motivos de Aminoácidos , Polaridade Celular , Células Epiteliais/virologia , Genoma Viral , Humanos , Transporte Proteico
6.
Virology ; 543: 20-26, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32056843

RESUMO

Human adenovirus serotype 7 (HAdV-7), belonging to species B, has caused severe lower respiratory tract diseases and even deaths recently. However, no adenovirus vaccine or therapeutic is available thus far. In this study, a HAdV-7-specific human monoclonal antibody (HMAb), 3-3E, isolated from single plasma cells obtained from the peripheral blood mononuclear cells of HAdV-7-infected patients showed potent HAdV-7 neutralization activity. The results showed HMAb 3-3E only binds to the hexon protein of intact HAdV-7 or the recombinant hexon protein and it does not bind to other intact virion particles. This could mean the antibody recognizes a conformational epitope of the hexon protein. Further, HMAb 3-3E potently neutralized HAdV-7 in vitro at low concentrations. In vivo studies showed HMAb 3-3E protected from HAdV-7 infection in a murine model. Therefore, HMAb 3-3E is promising as a safe and effective prophylactic and therapeutic treatment for HAdV-7 infection.


Assuntos
Infecções por Adenovirus Humanos/imunologia , Adenovírus Humanos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Vírion/imunologia , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Animais , Linhagem Celular , Mapeamento de Epitopos , Epitopos/imunologia , Expressão Gênica/genética , Humanos , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos SCID , Proteínas Recombinantes/genética , Sorogrupo , Vírion/genética , Vírion/metabolismo
7.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 12): 750-757, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31797817

RESUMO

The cryo-electron microscopy (cryo-EM) structure of the complex between the trimeric human adenovirus B serotype 3 fibre knob and human desmoglein 2 fragments containing cadherin domains EC2 and EC3 has been published, showing 3:1 and 3:2 complexes. Here, the crystal structure determined at 4.5 Šresolution is presented with one EC2-EC3 desmoglein fragment bound per fibre knob monomer in the asymmetric unit, leading to an apparent 3:3 stoichiometry. However, in concentrated solution the 3:2 complex is predominant, as shown by small-angle X-ray scattering (SAXS), while cryo-EM at lower concentrations showed a majority of the 3:1 complex. Substitution of the calcium ions bound to the desmoglein domains by terbium ions allowed confirmation of the X-ray model using their anomalous scattering and shows that at least one binding site per cluster of calcium ions is intact and exchangeable and, combined with SAXS data, that the cadherin domains are folded even in the distal part that is invisible in the cryo-EM reconstruction.


Assuntos
Adenovírus Humanos/metabolismo , Caderinas/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Desmogleína 2/química , Desmogleína 2/metabolismo , Adenovírus Humanos/classificação , Sequência de Aminoácidos , Caderinas/química , Cristalização , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Sorogrupo
8.
FEBS Lett ; 593(24): 3551-3570, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31769503

RESUMO

The DNA genome of eukaryotic cells is compacted by histone proteins within the nucleus to form chromatin. Nuclear-replicating viruses such as adenovirus have evolved mechanisms of chromatin manipulation to promote infection and subvert host defenses. Epigenetic factors may also regulate persistent adenovirus infection and reactivation in lymphoid tissues. In this review, we discuss the viral proteins E1A and protein VII that interact with and alter host chromatin, as well as E4orf3, which separates host chromatin from sites of viral replication. We also highlight recent advances in chromatin technologies that offer new insights into virus-directed chromatin manipulation. Beyond the role of chromatin in the viral replication cycle, we discuss the nature of persistent viral genomes in lymphoid tissue and cell lines, and the potential contribution of epigenetic signals in maintaining adenovirus in a quiescent state. By understanding the mechanisms through which adenovirus manipulates host chromatin, we will understand new aspects of this ubiquitous virus and shed light on previously unknown aspects of chromatin biology.


Assuntos
Infecções por Adenovirus Humanos/metabolismo , Adenovírus Humanos/patogenicidade , Cromatina/virologia , Epigênese Genética , Proteínas E1A de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/metabolismo , Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Cromatina/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Replicação Viral
9.
Virology ; 538: 24-34, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31561058

RESUMO

Human adenovirus (HAdV) can cause severe disease and death in both immunocompromised and immunocompetent patients. The current standards of treatment are often ineffective, and no approved antiviral therapy against HAdV exists. We report here the design and validation of a fluorescence-based high-content screening platform for the identification of novel anti-HAdV compounds. The screen was conducted using a wildtype-like virus containing the red fluorescent protein (RFP) gene under the regulation of the HAdV major late promoter. Thus, RFP expression allows monitoring of viral late gene expression (a surrogate marker for virus replication), and compounds affecting virus growth can be easily discovered by quantifying RFP intensity. We used our platform to screen ~1200 FDA-approved small molecules, and identified several cardiotonic steroids, corticosteroids and chemotherapeutic agents as anti-HAdV compounds. Our screening platform provides the stringency necessary to detect compounds with varying degrees of antiviral activity, and facilitates drug discovery/repurposing to combat HAdV infections.


Assuntos
Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/efeitos dos fármacos , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
10.
Sci Adv ; 5(9): eaax3567, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31517055

RESUMO

Adenoviruses are clinically important agents. They cause respiratory distress, gastroenteritis, and epidemic keratoconjunctivitis. As non-enveloped, double-stranded DNA viruses, they are easily manipulated, making them popular vectors for therapeutic applications, including vaccines. Species D adenovirus type 26 (HAdV-D26) is both a cause of EKC and other diseases and a promising vaccine vector. HAdV-D26-derived vaccines are under investigation as protective platforms against HIV, Zika, and respiratory syncytial virus infections and are in phase 3 clinical trials for Ebola. We recently demonstrated that HAdV-D26 does not use CD46 or Desmoglein-2 as entry receptors, while the putative interaction with coxsackie and adenovirus receptor is low affinity and unlikely to represent the primary cell receptor. Here, we establish sialic acid as a primary entry receptor used by HAdV-D26. We demonstrate that removal of cell surface sialic acid inhibits HAdV-D26 infection, and provide a high-resolution crystal structure of HAdV-D26 fiber-knob in complex with sialic acid.


Assuntos
Adenovírus Humanos/química , Ácido N-Acetilneuramínico/química , Receptores Virais/química , Proteínas Virais/química , Vacinas contra Adenovirus/metabolismo , Adenovírus Humanos/metabolismo , Cristalografia por Raios X , Células HEK293 , Humanos , Ceratoconjuntivite/epidemiologia , Ceratoconjuntivite/metabolismo , Ceratoconjuntivite/patologia , Ácido N-Acetilneuramínico/metabolismo , Receptores Virais/metabolismo , Proteínas Virais/metabolismo
11.
Vopr Virusol ; 64(2): 53-62, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31412171

RESUMO

Human adenoviruses cause different organ infections of varying severity, from asymptomatic to severe cases with lethal outcome, that are registered everywhere. Detailed and focused study of factors predisposing to a severe course of infection is required. The literature contains information indicating the association of severe adenoviral respiratory diseases with certain types of adenovirus, primarily type 7. This review highlights the possible causes of increased pathogenicity of some types of adenovirus and their association with severe forms of infection. Pathogenicity factors include the ability of adenovirus to bind the specific cellular receptors, the formation of subviral particles, the interaction with blood proteins, in particular the coagulation factor X, as well as the features of the early genes E1A, E1B, E3, E4. In addition, the severity of the disease may be affected by the presence or absence of pre-existing antibodies specific to certain types of adenoviruses. Chronic diseases or immunosuppression also increase the risk of severe adenovirus infection. The information presented in this review may elucidate the pathogenesis of adenovirus infection, and help to develop new features for prevention and treatment.


Assuntos
Infecções por Adenovirus Humanos , Adenovírus Humanos , Regulação Viral da Expressão Gênica , Infecções Respiratórias , Infecções por Adenovirus Humanos/genética , Infecções por Adenovirus Humanos/metabolismo , Infecções por Adenovirus Humanos/patologia , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Adenovírus Humanos/patogenicidade , Fator X/metabolismo , Humanos , Infecções Respiratórias/genética , Infecções Respiratórias/metabolismo , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
12.
Virology ; 536: 20-26, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31394408

RESUMO

The Coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and cell adhesion protein. CAR has two transmembrane isoforms that localize distinctly in polarized epithelial cells. Whereas the seven exon-encoded isoform (CAREx7) exhibits basolateral localization, the eight exon-encoded isoform (CAREx8) can localize to the apical epithelial surface where it can mediate luminal adenovirus infection. To further understand the distinct biological functions of these two isoforms, CRISPR/Cas9 genomic editing was used to specifically delete the eighth exon of the CXADR gene in a Madine Darby Canine Kidney (MDCK) cell line with a stably integrated lentiviral doxycycline-inducible CAREx8 cDNA. The gene-edited clone demonstrated a significant reduction in adenovirus susceptibility when both partially and fully polarized, and doxycycline-induction of CAREx8 restored sensitivity to adenovirus. These data reinforce the importance of CAREx8 in apical adenovirus infection and provide a new model cell line to probe isoform specific biological functions of CAR.


Assuntos
Adenovírus Humanos/genética , Sistemas CRISPR-Cas , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Edição de Genes/métodos , Regulação Viral da Expressão Gênica , Adenovírus Humanos/metabolismo , Animais , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Cães , Doxiciclina/farmacologia , Éxons , Humanos , Células Madin Darby de Rim Canino , Regiões Promotoras Genéticas/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Guia/genética , RNA Guia/metabolismo
13.
Cancer Lett ; 459: 15-29, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31150821

RESUMO

Pancreatic cancer is a highly lethal disease. Excessive accumulation of tumor extracellular matrix (ECM) and epithelial-to-mesenchymal transition (EMT) phenotype are two main contributors to drug resistance in desmoplastic pancreatic tumors. To overcome desmoplasia and chemoresistance of pancreatic cancer, we utilized an oncolytic adenovirus (Ad) co-expressing decorin and soluble Wnt decoy receptor (HEmT-DCN/sLRP6). An orthotopic pancreatic xenograft tumor model was established in athymic nude mice using Mia PaCa-2 cells, and the antimetastatic and antitumor efficacy of systemically administered HEmT-DCN/sLRP6 was evaluated. Immunohistochemical analysis of tumor tissues was performed to assess ECM degradation, induction of apoptosis, viral dispersion, and inhibition of the Wnt/ß-catenin signaling pathway. HEmT-DCN/sLRP6 effectively degraded tumor ECM and inhibited EMT, leading to enhanced viral distribution, induction of apoptosis, and attenuation of tumor cell proliferation in tumor tissue. HEmT-DCN/sLRP6 prevented metastasis of pancreatic cancer. Importantly, HEmT-DCN/sLRP6 sensitized pancreatic tumor to gemcitabine treatment. Furthermore, HEmT-DCN/sLRP6 augmented drug penetration and dispersion within pancreatic tumor xenografts and patient-derived tumor spheroids. Collectively, these results illustrate that HEmT-DCN/sLRP6 can enhance the dispersion of both oncolytic Ad and a chemotherapeutic agent in chemoresistant and desmoplastic pancreatic tumor, effectively overcoming the preexisting limitations of standard treatments.


Assuntos
Terapia Viral Oncolítica/métodos , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/virologia , Células A549 , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Animais , Linhagem Celular Tumoral , Decorina/biossíntese , Decorina/genética , Transição Epitelial-Mesenquimal , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/biossíntese , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Distribuição Aleatória , Receptores Wnt/antagonistas & inibidores , Receptores Wnt/metabolismo , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Pediatr Hematol Oncol ; 36(3): 161-172, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31037986

RESUMO

Human adenovirus (HAdV) is recognized as a serious pathogen after allogeneic hematopoietic stem cell transplantation (HSCT), causing morbidity and mortality. Currently, there is no universal agreement regarding routine HAdV surveillance after HSCT. We assessed the impact of HAdV weekly monitoring by polymerase chain reaction (PCR) on HAdV viremia rates and the risk factors that influence survival. Three-hundred and fifty-six pediatric allogeneic HSCT were done between 2007 and 2015. Until July 2011, HAdV testing was performed based on clinical suspicion (cohort 1, n = 175) and from August 2011, weekly blood-HAdV monitoring was done (cohort 2, n = 181) until day +100. Twenty-three patients (4 [2.3%] from cohort 1 and 19 [10.5%] from cohort 2, p = .001) were found with HAdV viremia and seven of them died. Both cohorts had a similar incidence of HAdV-associated mortality (3/175; 1.7% in cohort 1 and 4/181; 2.2% in cohort 2). Respiratory failure was the cause of death in all patients. Clinical symptoms appeared prior to or within 5 days of HAdV detection in cohort 2. In summary, weekly monitoring was associated with higher detection of HAdV. The study could not assess survival benefit due to small numbers of HAdV-positive cases. In many instances, symptoms occurred with the development of positive HAdV blood PCR results and hence, symptomatology could have triggered the test. Future studies are needed to provide data that help establishing a uniform approach for regular monitoring of HAdV post-transplant.


Assuntos
Infecções por Adenoviridae , Adenovírus Humanos , DNA Viral , Neoplasias Hematológicas , Transplante de Células-Tronco Hematopoéticas , Infecções por Adenoviridae/sangue , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/mortalidade , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , DNA Viral/sangue , DNA Viral/genética , Feminino , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/mortalidade , Neoplasias Hematológicas/terapia , Humanos , Lactente , Masculino , Fatores de Risco , Viremia/sangue , Viremia/genética , Viremia/mortalidade
15.
Nat Commun ; 10(1): 1181, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862836

RESUMO

Attachment of human adenovirus (HAd) to the host cell is a critical step of infection. Initial attachment occurs via the adenoviral fibre knob protein and a cellular receptor. Here we report the cryo-electron microscopy (cryo-EM) structure of a <100 kDa non-symmetrical complex comprising the trimeric HAd type 3 fibre knob (HAd3K) and human desmoglein 2 (DSG2). The structure reveals a unique stoichiometry of 1:1 and 2:1 (DSG2: knob trimer) not previously observed for other HAd-receptor complexes. We demonstrate that mutating Asp261 in the fibre knob is sufficient to totally abolish receptor binding. These data shed new light on adenovirus infection strategies and provide insights for adenoviral vector development and structure-based design.


Assuntos
Adenovírus Humanos/metabolismo , Proteínas do Capsídeo/metabolismo , Desmogleína 2/metabolismo , Receptores Virais/metabolismo , Ligação Viral , Infecções por Adenoviridae/patologia , Infecções por Adenoviridae/virologia , Adenovírus Humanos/patogenicidade , Asparagina/genética , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Desmogleína 2/ultraestrutura , Células HEK293 , Humanos , Modelos Moleculares , Domínios Proteicos , Receptores Virais/ultraestrutura , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
16.
Nat Commun ; 10(1): 741, 2019 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-30765704

RESUMO

Adenovirus based vectors are of increasing importance for wide ranging therapeutic applications. As vaccines, vectors derived from human adenovirus species D serotypes 26 and 48 (HAdV-D26/48) are demonstrating promising efficacy as protective platforms against infectious diseases. Significant clinical progress has been made, yet definitive studies underpinning mechanisms of entry, infection, and receptor usage are currently lacking. Here, we perform structural and biological analysis of the receptor binding fiber-knob protein of HAdV-D26/48, reporting crystal structures, and modelling putative interactions with two previously suggested attachment receptors, CD46 and Coxsackie and Adenovirus Receptor (CAR). We provide evidence of a low affinity interaction with CAR, with modelling suggesting affinity is attenuated through extended, semi-flexible loop structures, providing steric hindrance. Conversely, in silico and in vitro experiments are unable to provide evidence of interaction between HAdV-D26/48 fiber-knob with CD46, or with Desmoglein 2. Our findings provide insight into the cell-virus interactions of HAdV-D26/48, with important implications for the design and engineering of optimised Ad-based therapeutics.


Assuntos
Infecções por Adenoviridae/metabolismo , Adenovírus Humanos/metabolismo , Proteínas do Capsídeo/metabolismo , Receptores Virais/metabolismo , Infecções por Adenoviridae/virologia , Adenovírus Humanos/genética , Adenovírus Humanos/fisiologia , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/classificação , 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 , Variação Genética , Humanos , Proteína Cofatora de Membrana/química , Proteína Cofatora de Membrana/metabolismo , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , Receptores Virais/química , Homologia de Sequência de Aminoácidos
17.
Food Environ Virol ; 11(2): 178-183, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30747345

RESUMO

Although the effects of heavy metals on the behavior, including infectivity, of bacteria have been studied, little information is available about their effects on enteric viruses. We report an investigation of effects on the biosynthesis of human adenoviruses (HAdV) and hepatitis A (HAV) of waters contaminated with mineral waste following an environmental disaster in Mariana City, Minas Gerais State, Brazil. The study area was affected on November 5, 2015, by 60 million m3 of mud (containing very high concentrations of iron salts) from a mining reservoir (Fundão), reaching the Gualaxo do Norte River (sites evaluated in this study), the "Rio Doce" River and finally the Atlantic Ocean. We found substantial counts of infectious HAdV and HAV (by qPCR) in all sampled sites from Gualaxo do Norte River, indicating poor basic sanitation in this area. The effects of iron on viral infection processes were evaluated using HAdV-2 and HAV-175, as DNA and RNA enteric virus models, respectively, propagated in the laboratory and exposed to this contaminated water. Experiments in field and laboratory scales found that the numbers of plaque forming units (PFU) of HAdV and HAV were significantly higher in contaminated water with high iron concentrations than in waters with low iron concentration (< 20 µg/L of iron). These findings indicate that iron can potentiate enteric virus infectivity, posing a potential risk to human and animal health, particularly during pollution disasters such as that described here in Mariana, Brazil.


Assuntos
Adenovírus Humanos/crescimento & desenvolvimento , Vírus da Hepatite A/crescimento & desenvolvimento , Ferro/análise , Minerais/análise , Rios/virologia , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/genética , Adenovírus Humanos/isolamento & purificação , Adenovírus Humanos/metabolismo , Brasil , Enterovirus/genética , Enterovirus/crescimento & desenvolvimento , Enterovirus/isolamento & purificação , Enterovirus/metabolismo , Monitoramento Ambiental , Hepatite A/virologia , Vírus da Hepatite A/genética , Vírus da Hepatite A/isolamento & purificação , Vírus da Hepatite A/metabolismo , Humanos , Ferro/metabolismo , Mineração , Rios/química , Poluição da Água
18.
PLoS One ; 13(12): e0208427, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30521580

RESUMO

BACKGROUND: Type 2 diabetes is often linked with impaired proximal insulin signaling. Hence, a therapeutic agent that enhances cellular glucose uptake without requiring proximal insulin signaling would be desirable for improving glycemic control. The E4orf1 peptide (E4) derived from human adenovirus 36 (Ad36) promotes cellular glucose uptake in vitro and in vivo, independent of insulin. E4 bypasses a part of insulin signaling to upregulate cellular glucose uptake. We tested the hypothesis that E4 requires the distal but not proximal insulin signaling to enhance cellular glucose disposal. METHODS: 3T3-L1 preadipocytes inducibly expressing E4 or a null vector (NV) were treated with inhibitor of insulin receptor (S961), inhibitor of insulin like growth factor-1receptor (IGF-1R) (Picropodophyllin, PPP), PPP+S961, or phosphatidyl inositol-3 kinase (PI3K) inhibitor (Wortmannin, WM). We used PPP and S961 to block the proximal insulin signaling, or WM to block the distal insulin signaling. Cells were exposed to 0 or 100nM insulin. RESULTS: As expected, when the proximal or distal insulin signaling was blocked in NV cells, insulin could not enhance pAKT protein abundance, Glut4 translocation, or glucose uptake. Whereas, E4 cells significantly increased pAKT abundance, Glut4 translocation and glucose uptake independent of the presence of insulin or proximal insulin signaling. Enhanced glucose disposal in E4 cells was completely abrogated when the distal insulin signaling was blocked. CONCLUSIONS: E4 bypasses the proximal insulin signaling but uses the distal insulin signaling to activate pAkt and in turn Glut4 translocation to improve cellular glucose uptake. E4 offers a promising template to improve glycemic control when the proximal insulin signaling is impaired.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Virais/farmacologia , Células 3T3-L1 , Adenovírus Humanos/metabolismo , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Transportador de Glucose Tipo 4/metabolismo , Camundongos , Modelos Biológicos , Peptídeos/farmacologia , Fosforilação , Podofilotoxina/análogos & derivados , Podofilotoxina/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Wortmanina/farmacologia
19.
Biochim Biophys Acta Biomembr ; 1860(11): 2215-2223, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30409517

RESUMO

Adenoviral dodecahedron is a virus-like particle composed of twelve penton base proteins, derived from the capsid of human adenovirus type 3. Due to the high cell penetration capacity, it was used as a vector for protein, peptide and drug delivery. Two receptors are known to be involved in the endocytic dodecahedron uptake, namely αv integrins and heparan sulfate proteoglycans. Since it has been observed, that dodecahedron efficiently penetrates a wide range of cancer cells, it suggests that other cellular compounds may play a role in the particle endocytosis. To shed some light onto the interactions with membrane lipids and their potential role in dodecahedron entry, we performed a series of experiments including biochemical assays, fluorescence confocal imaging of giant unilamellar vesicles and surface plasmon resonance, which indicated specific preference of the particle to anionic phosphatidylserine. Experiments performed on cholesterol-depleted epithelial cells showed that cholesterol is essential in the endocytic uptake, however a direct interaction was not observed. We believe that the results will allow to better understand the role of lipids in dodecahedron entry and to design more specific dodecahedron-based vectors for drug delivery to cancer cells.


Assuntos
Adenovírus Humanos/metabolismo , Colesterol/metabolismo , Endocitose , Fosfatidilserinas/metabolismo , Anexina A5/metabolismo , Células HeLa , Humanos , Lipídeos de Membrana/metabolismo , Ressonância de Plasmônio de Superfície
20.
PLoS One ; 13(9): e0204522, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30252905

RESUMO

Viral infections cause large problems in the world and deeper understanding of the disease mechanisms is needed. Here we present an analytical strategy to investigate the host cell protein changes during human adenovirus type 2 (HAdV-C2 or Ad2) infection of lung fibroblasts by stable isotope labelling of amino acids in cell culture (SILAC) and nanoLC-MS/MS. This work focuses on early phase of infection (6 and 12 h post-infection (hpi)) but the data is combined with previously published late phase (24 and 36 hpi) proteomics data to produce a time series covering the complete infection. As many as 2169 proteins were quantitatively monitored from 6 to 36 hpi, while some proteins were time-specific. After applying different filter criteria, 2027 and 2150 proteins were quantified at 6 and 12 hpi and among them, 431 and 544 were significantly altered at the two time points. Pathway analysis showed that the De novo purine and pyrimidine biosynthesis, Glycolysis and Cytoskeletal regulation by Rho GTPase pathways were activated early during infection while inactivation of the Integrin signalling pathway started between 6 and 12 hpi. Moreover, upstream regulator analysis predicted MYC to be activated with time of infection and protein and RNA data for genes controlled by this transcription factor showed good correlation, which validated the use of protein data for this prediction. Among the identified phosphorylation sites, a group related to glycolysis and cytoskeletal reorganization were up-regulated during infection. The results show specific aspects on how the host cell proteins, the final products in the genetic information flow, are influenced by Ad2 infection, which would be overlooked if only knowledge derived from mRNA data is considered.


Assuntos
Infecções por Adenoviridae/metabolismo , Adenovírus Humanos/metabolismo , Infecções por Adenoviridae/genética , Infecções por Adenoviridae/virologia , Adenovírus Humanos/genética , Aminoácidos/metabolismo , Linhagem Celular , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Cinética , Redes e Vias Metabólicas/genética , Fosfopeptídeos/genética , Fosfopeptídeos/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espectrometria de Massas em Tandem , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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