38 research outputs found
The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis coronavirus
<p>Abstract</p> <p>Background</p> <p>Transmissible gastroenteritis virus (TGEV) has a sialic acid binding activity that is believed to be important for enteropathogenicity, but that has so far appeared to be dispensable for infection of cultured cells. The aims of this study were to determine the effect of sialic acid binding for the infection of cultured cells under unfavorable conditions, and comparison of TGEV strains and mutants, as well as the avian coronavirus IBV concerning their dependence on the sialic acid binding activity.</p> <p>Methods</p> <p>The infectivity of different viruses was analyzed by a plaque assay after adsorption times of 5, 20, and 60 min. Prior to infection, cultured cells were either treated with neuraminidase to deplete sialic acids from the cell surface, or mock-treated. In a second approach, pre-treatment of the virus with porcine intestinal mucin was performed, followed by the plaque assay after a 5 min adsorption time. A student's t-test was used to verify the significance of the results.</p> <p>Results</p> <p>Desialylation of cells only had a minor effect on the infection by TGEV strain Purdue 46 when an adsorption period of 60 min was allowed for initiation of infection. However, when the adsorption time was reduced to 5 min the infectivity on desialylated cells decreased by more than 60%. A TGEV PUR46 mutant (HAD3) deficient in sialic acid binding showed a 77% lower titer than the parental virus after a 5 min adsorption time. After an adsorption time of 60 min the titer of HAD3 was 58% lower than that of TGEV PUR46. Another TGEV strain, TGEV Miller, and IBV Beaudette showed a reduction in infectivity after neuraminidase treatment of the cultured cells irrespective of the virion adsorption time.</p> <p>Conclusions</p> <p>Our results suggest that the sialic acid binding activity facilitates the infection by TGEV under unfavorable environmental conditions. The dependence on the sialic acid binding activity for an efficient infection differs in the analyzed TGEV strains.</p
Characterization of the sialic acid binding activity of Influenza A viruses using soluble variants of the H7 and H9 hemagglutinins
Binding of influenza viruses to target cells is mediated by the viral surface protein hemagglutinin. To determine the presence of binding sites for influenza A viruses on cells and tissues, soluble hemagglutinins of the H7 and H9 subtype were generated by connecting the hemagglutinin ectodomain to the Fc portion of human immunoglobulin G (H7Fc and H9Fc). Both chimeric proteins bound to different cells and tissues in a sialic acid-dependent manner. Pronounced differences were observed between H7Fc and H9Fc, in the binding both to different mammalian and avian cultured cells and to cryosections of the respiratory epithelium of different virus host species (turkey, chicken and pig). Binding of the soluble hemagglutinins was similar to the binding of virus particles, but showed differences in the binding pattern when compared to two sialic acid-specific plant lectins. These findings were substantiated by a comparative glycan array analysis revealing a very narrow recognition of sialoglycoconjugates by the plant lectins that does not reflect the glycan structures preferentially recognized by H7Fc and H9Fc. Thus, soluble hemagglutinins may serve as sialic acid-specific lectins and are a more reliable indicator of the presence of binding sites for influenza virus HA than the commonly used plant lectins
The SARS-coronavirus-host interactome
Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock
The SARS-Coronavirus-Host Interactome: Identification of Cyclophilins as Target for Pan-Coronavirus Inhibitors
Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock
Infection of Differentiated Porcine Airway Epithelial Cells by Influenza Virus: Differential Susceptibility to Infection by Porcine and Avian Viruses
BACKGROUND: Swine are important hosts for influenza A viruses playing a crucial role in the epidemiology and interspecies transmission of these viruses. Respiratory epithelial cells are the primary target cells for influenza viruses. METHODOLOGY/PRINCIPAL FINDINGS: To analyze the infection of porcine airway epithelial cells by influenza viruses, we established precision-cut lung slices as a culture system for differentiated respiratory epithelial cells. Both ciliated and mucus-producing cells were found to be susceptible to infection by swine influenza A virus (H3N2 subtype) with high titers of infectious virus released into the supernatant already one day after infection. By comparison, growth of two avian influenza viruses (subtypes H9N2 and H7N7) was delayed by about 24 h. The two avian viruses differed both in the spectrum of susceptible cells and in the efficiency of replication. As the H9N2 virus grew to titers that were only tenfold lower than that of a porcine H3N2 virus this avian virus is an interesting candidate for interspecies transmission. Lectin staining indicated the presence of both α-2,3- and α-2,6-linked sialic acids on airway epithelial cells. However, their distribution did not correlate with pattern of virus infection indicating that staining by plant lectins is not a reliable indicator for the presence of cellular receptors for influenza viruses. CONCLUSIONS/SIGNIFICANCE: Differentiated respiratory epithelial cells significantly differ in their susceptibility to infection by avian influenza viruses. We expect that the newly described precision-cut lung slices from the swine lung are an interesting culture system to analyze the infection of differentiated respiratory epithelial cells by different pathogens (viral, bacterial and parasitic ones) of swine
The Spike Protein of Infectious Bronchitis Virus Is Retained Intracellularly by a Tyrosine Motif▿
We have analyzed the intracellular transport of the spike (S) protein of infectious bronchitis virus (IBV), an avian coronavirus. Surface expression was analyzed by immunofluorescence microscopy, by surface biotinylation, and by syncytium formation by S-expressing cells. By applying these methods, the S protein was found to be retained intracellularly. Tyr1143 in the cytoplasmic tail was shown to be a crucial component of the retention signal. Deletion of a dilysine motif that has previously been suggested to function as a retrieval signal did not abolish intracellular retention. Treatment of the S proteins with endoglycosidases did not reveal any differences between the parental and the mutant proteins. Furthermore, all S proteins analyzed were posttranslationally cleaved into the subunits S1 and S2. In coexpression experiments, the S protein was found to colocalize with a Golgi marker. Taken together, these results indicate that the S protein of IBV is retained at a late Golgi compartment. Therefore, this viral surface protein differs from the S proteins of transmissible gastroenteritis virus and severe acute respiratory syndrome coronavirus, which are retained at a pre-Golgi compartment or transported to the cell surface, respectively. The implications of these differences are discussed
Cholesterol is important for a post-adsorption step in the entry process of transmissible gastroenteritis virus
Cholesterol is a major constituent of detergent-resistant membrane microdomains (DRMs). We localized transmissible gastroenteritis virus (TGEV) spike (S) protein in DRMs in the viral envelope. Though S protein was not solubilized by cold non-ionic detergents, this behavior was unchanged when cholesterol was depleted from viral membrane by methyl-β-cyclodextrin (MβCD) and the protein did not comigrate with cellular DRM marker proteins in flotation analyses. Therefore, the S protein is not anchored in the viral membrane DRMs as they are known to occur in the plasma membrane. Cholesterol depletion from viral membrane may not affect the adsorption process as neither the sialic acid binding activity nor the binding to aminopeptidase N was reduced post-MβCD treatment. Reduced infectivity of cholesterol-depleted TGEV was observed only when the adsorption process occurred at 37 °C but not when the virus was applied at 4 °C. Cholesterol is important for a post-adsorption step, allowing membrane rearrangements that facilitate virus entry. © 2010 Elsevier B.V.National Natural Science Foundation of China (30700591) to J.Y. National Natural Science Foundation of China (30700590; 30972195), Funding supported by Program for New Century Excellent Talents in Heilongjiang Provincial University (1155--NCET--005) and a visiting scholarship from Deutscher Akademischer Austauschdienst (DAAD) to X.R. are acknowledged. G.H. was supported by Deutsche Forschungsgemeinschaft (SFB621) and by the German Ministry of Education and Research (BMBF: FKZ 01KI0702
Infection of porcine precision cut intestinal slices by transmissible gastroenteritis coronavirus demonstrates the importance of the spike protein for enterotropism of different virus strains
Comparison of vesicular stomatitis virus pseudotyped with the S proteins from a porcine and a human coronavirus
The surface proteins S of severe acute respiratory syndrome coronavirus (SARS-CoV) and transmissible gastroenteritis virus (TGEV) were compared for their ability to mediate infection of viral pseudotypes based on vesicular stomatitis virus (VSV). The cell tropism of the respective pseudotypes corresponded to the tropism of the viruses from which the S protein was derived. Higher infectivity values were obtained with the SARS-CoV S protein than with the TGEV S protein. Differences were observed with respect to the importance of the cytoplasmic tail and the membrane anchor of the S proteins. In the case of the SARS-CoV S protein, truncation of the cytoplasmic tail resulted in increased infectivity. For the TGEV S protein, the inactivation of an intracellular retention signal in the cytoplasmic tail was required. Exchange of the membrane anchor of the S proteins led to a low infection efficiency. Our results indicate that related glycoproteins may show substantial differences in their ability to mediate pseudotype infection. © 2009 SGM.This work was supported by grants to G. H. from Deutsche Forschungsgemeinschaft (SFB621), the German Ministry of Education and Research (project code ‘O¨ kologie und Pathogenese von SARS’), and the European Union