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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
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
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.
Human adenovirus (HAdV)-F40 and -F41 are leading causes of diarrhea and diarrhea-associated mortality in children under the age of five, but the mechanisms by which they infect host cells are poorly ...understood. HAdVs initiate infection through interactions between the knob domain of the fiber capsid protein and host cell receptors. Unlike most other HAdVs, HAdV-F40 and -F41 possess two different fiber proteins-a long fiber and a short fiber. Whereas the long fiber binds to the Coxsackievirus and adenovirus receptor (CAR), no binding partners have been identified for the short fiber. In this study, we identified heparan sulfate (HS) as an interaction partner for the short fiber of enteric HAdVs. We demonstrate that exposure to acidic pH, which mimics the environment of the stomach, inactivates the interaction of enteric adenovirus with CAR. However, the short fiber:HS interaction is resistant to and even enhanced by acidic pH, which allows attachment to host cells. Our results suggest a switch in receptor usage of enteric HAdVs after exposure to acidic pH and add to the understanding of the function of the short fibers. These results may also be useful for antiviral drug development and the utilization of enteric HAdVs for clinical applications such as vaccine development.
Virus entry into host cells is a complex process that is largely regulated by the 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 raise the question how adenovirus - 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 map this effect to the proteolytically cleavable, positively charged, N-terminal 49 residues of human lactoferrin (hLF) known as lactoferricin (hLfcin). Lfcin binds to the hexon protein on the viral capsid, and anchors the virus to an unknown receptor of target cells, resulting in infection. These findings suggests 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, and when infection is established, progeny virions released from the basolateral side enter neighbouring cells by means of hLF/hLfcin and CAR in parallel.
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.
The coronavirus disease 2019, COVID-19, is a complex disease with a wide range of symptoms from asymptomatic infections to severe acute respiratory syndrome with lethal outcome. Individual factors ...such as age, sex, and comorbidities increase the risk for severe infections, but other aspects, such as genetic variations, are also likely to affect the susceptibility to SARS-CoV-2 infection and disease severity. Here, we used a human 3D lung cell model based on primary cells derived from multiple donors to identity host factors that regulate SARS-CoV-2 infection. With a transcriptomics-based approach, we found that less susceptible donors show a higher expression level of serine protease inhibitors SERPINA1, SERPINE1, and SERPINE2, identifying variation in cellular serpin levels as restricting host factors for SARS-CoV-2 infection. We pinpoint their antiviral mechanism of action to inhibition of the cellular serine protease, TMPRSS2, thereby preventing cleavage of the viral spike protein and TMPRSS2-mediated entry into the target cells. By means of single-cell RNA sequencing, we further locate the expression of the individual serpins to basal, ciliated, club, and goblet cells. Our results add to the importance of genetic variations as determinants for SARS-CoV-2 susceptibility and suggest that genetic deficiencies of cellular serpins might represent risk factors for severe COVID-19. Our study further highlights TMPRSS2 as a promising target for antiviral intervention and opens the door for the usage of locally administered serpins as a treatment against COVID-19.
Identification of host factors affecting individual SARS-CoV-2 susceptibility will provide a better understanding of the large variations in disease severity and will identify potential factors that can be used, or targeted, in antiviral drug development. With the use of an advanced lung cell model established from several human donors, we identified cellular protease inhibitors, serpins, as host factors that restrict SARS-CoV-2 infection. The antiviral mechanism was found to be mediated by the inhibition of a serine protease, TMPRSS2, which results in a blockage of viral entry into target cells. Potential treatments with these serpins would not only reduce the overall viral burden in the patients, but also block the infection at an early time point, reducing the risk for the hyperactive immune response common in patients with severe COVID-19.
Bacterial kidney disease (BKD) can be a devastating bacterial infection in salmonids, and it is present in aquaculture throughout the world. BKD is caused by the Gram‐positive facultative ...intracellular bacterium Renibacterium salmoninarum (R. salmoninarum) that is spread both horizontally and vertically. Disease signs include external ulcerations and blisters and internal signs such as organ swelling, granulomas, petechiae and ascites. In Sweden, BKD accounts for a significant income loss in aquacultures due to expensive decontamination of the facility and increased disease susceptibility for the immunocompromised fish leading to higher mortality rates. In addition, uncontrolled spread in aquaculture may threaten the survival of wild fish populations. The aim of our study was to investigate the prevalence of R. salmoninarum in wild salmonids caught in Swedish waters where net pen farms with a recent history of BKD are present. Four rivers with at least one BKD‐positive or recently BKD‐positive farm were selected. In addition, we evaluated the use of environmental DNA (eDNA) for surveillance and monitoring of ongoing infections at these locations. In total, 1058 fish were sampled from four different river systems, and of them 52 (4.9%) were positive for R. salmoninarum by antigen ELISA. Surprisingly, these fish were not evenly distributed between the four river systems, but 50 were caught in the same river (Ljungan). This accounts for an alarmingly high rate of 17% R. salmoninarum‐positive samples in wild salmonids in this area. This number is far above what was expected and clearly shows the risk with an open farming system as well as the importance of effective health monitoring programmes to avoid an uncontrolled spread of the disease. The use of eDNA for monitoring BKD is somewhat difficult to evaluate. Few of the water samples analysed were PCR positive for R. salmoninarum (2 of 38) and those were collected where no ELISA positive fish were identified. In addition to water, sediment samples were collected under a net pen farm that had recently slaughtered all fish due to ongoing R. salmoninarum infections. Sediment samples are more promising than water as 4 of 5 samples at one farming facility where positive for R. salmoninarum. Thus, sediment samples may be valuable for monitoring potential ongoing BKD in farms, without the need to sacrifice valuable fish.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The heterocoupling of organic building blocks to give complex multicomponent macromolecules directly at a surface holds the key to creating advanced molecular devices. While “on-surface” synthesis ...with prefunctionalized molecules has recently led to specific one- and two- component products, a central challenge is to discover universal connection strategies that are applicable to a wide range of molecules. Here, we show that direct activation of C–H bonds intrinsic to π-functional molecules is a highly generic route for connecting different building blocks on a copper surface. Scanning tunneling microscopy (STM) reveals that covalent π-functional macromolecular heterostructures, displaying diverse compositions, structures and topologies, are created with ease from seven distinct building blocks (including porphyrins, pentacene and perylene). By exploiting differences in C–H bond reactivity in the deposition and heating protocols we also demonstrate controlled synthesis of specific products, such as block copolymers. Further, the symmetry and geometry of the molecules and the surface also play a critical role in determining the outcome of the covalent bond forming reactions. Our “pick-mix-and-link” strategy opens up the capability to generate libraries of multivariate macromolecules directly at a surface, which in conjunction with nanoscale probing techniques could accelerate the discovery of functional interfaces.
Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment ...and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily
its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development.
Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.
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