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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(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.
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
4.
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
5.
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
6.
PLoS One ; 9(7): e102368, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25033192

RESUMO

Respiratory syncytial virus (RSV) is a major cause of morbidity and mortality worldwide, causing severe respiratory illness in infants and immune compromised patients. The ciliated cells of the human airway epithelium have been considered to be the exclusive target of RSV, although recent data have suggested that basal cells, the progenitors for the conducting airway epithelium, may also become infected in vivo. Using either mechanical or chemical injury models, we have demonstrated a robust RSV infection of p63+ basal cells in air-liquid interface (ALI) cultures of human bronchial epithelial cells. In addition, proliferating basal cells in 2D culture were also susceptible to RSV infection. We therefore tested the hypothesis that RSV infection of this progenitor cell would influence the differentiation status of the airway epithelium. RSV infection of basal cells on the day of seeding (MOI≤0.0001), resulted in the formation of an epithelium that showed a profound loss of ciliated cells and gain of secretory cells as assessed by acetylated α-tubulin and MUC5AC/MUC5B immunostaining, respectively. The mechanism driving the switch in epithelial phenotype is in part driven by the induced type I and type III interferon response that we demonstrate is triggered early following RSV infection. Neutralization of this response attenuates the RSV-induced loss of ciliated cells. Together, these data show that through infection of proliferating airway basal cells, RSV has the potential to influence the cellular composition of the airway epithelium. The resulting phenotype might be expected to contribute towards both the severity of acute infection, as well as to the longer-term consequences of viral exacerbations in patients with pre-existing respiratory diseases.


Assuntos
Diferenciação Celular , Células Epiteliais/citologia , Mucosa Respiratória/virologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/patologia , Proliferação de Células , Células Cultivadas , Células Epiteliais/virologia , Humanos , Interferon Tipo I/imunologia , Interferons , Interleucinas/imunologia , Mucina-5AC/metabolismo , Mucina-5B/metabolismo , Mucosa Respiratória/citologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/imunologia , Vírus Sinciciais Respiratórios/patogenicidade , Células-Tronco/virologia , Tubulina (Proteína)/metabolismo
7.
PLoS Pathog ; 6(9): e1001122, 2010 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-20941397

RESUMO

The human membrane cofactor protein (MCP, CD46) is a central component of the innate immune system. CD46 protects autologous cells from complement attack by binding to complement proteins C3b and C4b and serving as a cofactor for their cleavage. Recent data show that CD46 also plays a role in mediating acquired immune responses, and in triggering autophagy. In addition to these physiologic functions, a significant number of pathogens, including select adenoviruses, measles virus, human herpes virus 6 (HHV-6), Streptococci, and Neisseria, use CD46 as a cell attachment receptor. We have determined the crystal structure of the extracellular region of CD46 in complex with the human adenovirus type 11 fiber knob. Extracellular CD46 comprises four short consensus repeats (SCR1-SCR4) that form an elongated structure resembling a hockey stick, with a long shaft and a short blade. Domains SCR1, SCR2 and SCR3 are arranged in a nearly linear fashion. Unexpectedly, however, the structure reveals a profound bend between domains SCR3 and SCR4, which has implications for the interactions with ligands as well as the orientation of the protein at the cell surface. This bend can be attributed to an insertion of five hydrophobic residues in a SCR3 surface loop. Residues in this loop have been implicated in interactions with complement, indicating that the bend participates in binding to C3b and C4b. The structure provides an accurate framework for mapping all known ligand binding sites onto the surface of CD46, thereby advancing an understanding of how CD46 acts as a receptor for pathogens and physiologic ligands of the immune system.


Assuntos
Adenovírus Humanos/metabolismo , Complemento C3b/metabolismo , Complemento C4b/metabolismo , Proteína Cofatora de Membrana/química , Proteína Cofatora de Membrana/metabolismo , Adenovírus Humanos/classificação , Sítios de Ligação , Cristalografia por Raios X , Glicosilação , Humanos , Modelos Moleculares , Polissacarídeos/metabolismo , Conformação Proteica , Receptores Virais/metabolismo
8.
J Virol ; 84(7): 3189-200, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20071571

RESUMO

The complement regulation protein CD46 is the primary attachment receptor for most species B adenoviruses (Ads). However, significant variability exists in sequence and structure among species B Ads in the CD46-binding regions, correlating with differences in affinity. Here, we report a structure-function analysis of the interaction of the species B Ad21 knob with the two N-terminal repeats SCR1 and SCR2 of CD46, CD46-D2. We have determined the structures of the Ad21 knob in its unliganded form as well as in complex with CD46-D2, and we compare the interactions with those observed for the Ad11 knob-CD46-D2 complex. Surface plasmon resonance measurements demonstrate that the affinity of Ad21 knobs for CD46-D2 is 22-fold lower than that of the Ad11 knob. The superposition of the Ad21 and Ad11 knob structures in complex with CD46-D2 reveals a substantially different binding mode, providing an explanation for the weaker binding affinity of the Ad21 knob for its receptor. A critical difference in both complex structures is that a key interaction point, the DG loop, protrudes more in the Ad21 knob than in the Ad11 knob. Therefore, the protruding DG loop does not allow CD46-D2 to approach the core of the Ad21 knob as closely as in the Ad11 knob-CD46-D2 complex. In addition, the engagement of CD46-D2 induces a conformational change in the DG loop in the Ad21 knob but not in the Ad11 knob. Our results contribute to a more profound understanding of the CD46-binding mechanism of species B Ads and have relevance for the design of more efficient gene delivery vectors.


Assuntos
Adenovírus Humanos/química , Proteínas do Capsídeo/química , Proteína Cofatora de Membrana/química , Receptores Virais/química , Ligação Viral , Adenovírus Humanos/classificação , Adenovírus Humanos/fisiologia , Sequência de Aminoácidos , Proteína Cofatora de Membrana/metabolismo , Dados de Sequência Molecular , Conformação Proteica , Ressonância de Plasmônio de Superfície
9.
J Virol ; 83(2): 673-86, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18987134

RESUMO

Adenoviruses (Ads) are icosahedral, nonenveloped viruses with a double-stranded DNA genome. The 51 known Ad serotypes exhibit profound variations in cell tropism and disease types. The number of observed Ad infections is steadily increasing, sometimes leading to fatal outcomes even in healthy individuals. Species B Ads can cause kidney infections, hemorrhagic cystitis, and severe respiratory infections, and most of them use the membrane cofactor protein CD46 as a cellular receptor. The crystal structure of the human Ad type 11 (Ad11) knob complexed with CD46 is known; however, the determinants of CD46 binding in related species B Ads remain unclear. We report here a structural and functional analysis of the Ad11 knob, as well as the Ad7 and Ad14 knobs, which are closely related in sequence to the Ad11 knob but have altered CD46-binding properties. The comparison of the structures of the three knobs, which we determined at very high resolution, provides a platform for understanding these differences and allows us to propose a mechanism for productive high-affinity engagement of CD46. At the center of this mechanism is an Ad knob arginine that needs to switch its orientation in order to engage CD46 with high affinity. Quantum chemical calculations showed that the CD46-binding affinity of Ad11 is significantly higher than that of Ad7. Thus, while Ad7 and Ad14 also bind CD46, the affinity and kinetics of these interactions suggest that these Ads are unlikely to use CD46 productively. The proposed mechanism is likely to determine the receptor usage of all CD46-binding Ads.


Assuntos
Adenoviridae/química , Adenoviridae/fisiologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Proteína Cofatora de Membrana/metabolismo , Ligação Viral , Cristalografia por Raios X , Humanos , Ligação Proteica , Estrutura Terciária de Proteína
10.
Nat Struct Mol Biol ; 14(2): 164-6, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17220899

RESUMO

Adenoviruses (Ads) are important human pathogens and valuable gene delivery vehicles. We report here the crystal structure of the species B Ad11 knob complexed with the Ad11-binding region of its receptor CD46. The conformation of bound CD46 differs profoundly from its unbound state, with the bent surface structure straightened into an elongated rod. This mechanism of interaction is likely to be conserved among many pathogens that target CD46 or related molecules.


Assuntos
Adenovírus Humanos/metabolismo , Receptores Virais/química , Proteínas Virais/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Receptores Virais/metabolismo , Proteínas Virais/metabolismo
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