Dynamic Innate Immune Responses of Human Bronchial Epithelial Cells to Severe Acute Respiratory Syndrome-Associated Coronavirus Infection

Human lung epithelial cells are likely among the first targets to encounter invading severe acute respiratory syndrome-associated coronavirus (SARS-CoV). Not only can these cells support the growth of SARS-CoV infection, but they are also capable of secreting inflammatory cytokines to initiate and, eventually, aggravate host innate inflammatory responses, causing detrimental immune-mediated pathology within the lungs. Thus, a comprehensive evaluation of the complex epithelial signaling to SARS-CoV is crucial for paving the way to better understand SARS pathogenesis. Based on microarray-based functional genomics, we report here the global gene response of 2B4 cells, a cloned bronchial epithelial cell line derived from Calu-3 cells. Specifically, we found a temporal and spatial activation of nuclear factor (NF)κB, activator protein (AP)-1, and interferon regulatory factor (IRF)-3/7 in infected 2B4 cells at 12-, 24-, and 48-hrs post infection (p.i.), resulting in the activation of many antiviral genes, including interferon (IFN)-β, -λs, inflammatory mediators, and many IFN-stimulated genes (ISGs). We also showed, for the first time, that IFN-β and IFN-λs were capable of exerting previously unrecognized, non-redundant, and complementary abilities to limit SARS-CoV replication, even though their expression could not be detected in infected 2B4 bronchial epithelial cells until 48 hrs p.i. Collectively, our results highlight the mechanics of the sequential events of antiviral signaling pathway/s triggered by SARS-CoV in bronchial epithelial cells and identify novel cellular targets for future studies, aiming at advancing strategies against SARS

Release of Severe Acute Respiratory Syndrome Coronavirus Nuclear Import Block Enhances Host Transcription in Human Lung Cells

The severe acute respiratory syndrome coronavirus accessory protein ORF6 antagonizes interferon signaling by blocking karyopherin-mediated nuclear import processes. Viral nuclear import antagonists, expressed by several highly pathogenic RNA viruses, likely mediate pleiotropic effects on host gene expression, presumably interfering with transcription factors, cytokines, hormones, and/or signaling cascades that occur in response to infection. By bioinformatic and systems biology approaches, we evaluated the impact of nuclear import antagonism on host expression networks by using human lung epithelial cells infected with either wild-type virus or a mutant that does not express ORF6 protein. Microarray analysis revealed significant changes in differential gene expression, with approximately twice as many upregulated genes in the mutant virus samples by 48 h postinfection, despite identical viral titers. Our data demonstrated that ORF6 protein expression attenuates the activity of numerous karyopherin-dependent host transcription factors (VDR, CREB1, SMAD4, p53, EpasI, and Oct3/4) that are critical for establishing antiviral responses and regulating key host responses during virus infection. Results were confirmed by proteomic and chromatin immunoprecipitation assay analyses and in parallel microarray studies using infected primary human airway epithelial cell cultures. The data strongly support the hypothesis that viral antagonists of nuclear import actively manipulate host responses in specific hierarchical patterns, contributing to the viral pathogenic potential in vivo. Importantly, these studies and modeling approaches not only provide templates for evaluating virus antagonism of nuclear import processes but also can reveal candidate cellular genes and pathways that may significantly influence disease outcomes following severe acute respiratory syndrome coronavirus infection in vivo

RIG-I enhanced interferon independent apoptosis upon Junin virus infection.

Junin virus (JUNV) is the etiological agent of Argentine hemorrhagic fever (AHF), a human disease with a high case-fatality rate. It is widely accepted that arenaviral infections, including JUNV infections, are generally non-cytopathic. In contrast, here we demonstrated apoptosis induction in human lung epithelial carcinoma (A549), human hepatocarcinoma and Vero cells upon infection with the attenuated Candid#1 strain of, JUNV as determined by phosphatidylserine (PS) translocation, Caspase 3 (CASP3) activation, Poly (ADP-ribose) polymerase (PARP) cleavage and/or chromosomal DNA fragmentation. Moreover, as determined by DNA fragmentation, we found that the pathogenic Romero strain of JUNV was less cytopathic than Candid#1 in human hepatocarcinoma and Vero, but more apoptotic in A549 and Vero E6 cells. Additionally, we found that JUNV-induced apoptosis was enhanced by RIG-I signaling. Consistent with the previously reported role of RIG-I like helicase (RLH) signaling in initiating programmed cell death, we showed that cell death or DNA fragmentation of Candid#1-infected A549 cells was decreased upon siRNA or shRNA silencing of components of RIG-I pathway in spite of increased virus production. Similarly, we observed decreased DNA fragmentation in JUNV-infected human hepatocarcinoma cells deficient for RIG-I when compared with that of RIG-I-competent cells. In addition, DNA fragmentation detected upon Candid#1 infection of type I interferon (IFN)-deficient Vero cells suggested a type I IFN-independent mechanism of apoptosis induction in response to JUNV. Our work demonstrated for the first time apoptosis induction in various cells of mammalian origin in response to JUNV infection and partial mechanism of this cell death

Temporal and differential activation of transcription factors (TFs) in SARS-CoV-infected 2B4 cells.

<p>Genes whose expressions were significantly altered in SARS-CoV-infected 2B4 cells were subjected to the TRANSFAC database-based analysis of TF activation. Adjusted <i>p</i> values of <0.05 among stringent pairwise comparisons were used for selecting those TFs that were significantly enriched at 12, 24, and 48 hrs p.i., respectively.</p

Efficacy of IFN-λs in the host defense against SARS-CoV infection.

<p>Confluent cultures of 2B4 cells were treated with rIFN-β, rIFN-λ1, or rIFN-λ2 at the indicated concentrations alone (A) or in combination (B) for 24 hrs prior to their infection with SARS-CoV (MOI = 0.01). Resulting supernatants were harvested at day 2 after infection for assessing yields of infectious progeny virus by the standard Vero E6-based infectivity assay. *, p<0.05 by a two-way ANOVA with Bonferroni's post-hoc test in comparison with IFN-untreated cultures. Data shown were representative of two independent experiments.</p

Enriched molecular functions of those genes whose expressions in 2B4 cells were significantly altered by SARS-CoV.

<p>Expressions of both up- and down-regulated genes at 12, 24, and/or 48 hrs after SARS-CoV infection were analyzed against the entire human genome gene set. The enriched GO-annotated terms identified for those up-regulated and down-regulated genes are presented in A and B, respectively. The height of an individual bar represents the level of the statistical significance of the enriched GO-annotated term. An adjusted <i>p</i> value of <0.05 was used as the criterion for selecting enriched molecular functions.</p

Characteristics of 2B4 cells clonally derived from human bronchial epithelial Calu-3 cells.

<p>Expressions of the viral ACE2 receptor in indicated passages of 2B4 cells and their parental Calu-3 cells were assessed by standard IHC (A) and Western blot analysis (B), whereas the morphological features of polarized 2B4 cells were assessed by TEM (C), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008729#s4" target="_blank">Materials and Methods</a>. The images were taken at 6,270 magnifications. The scale bar represents 1 µm. To compare the permissiveness of 2B4 cells to their parental Calu-3 cells, confluent 2B4 cells, at passages #6 and #12, and Calu-3 cells were subjected to SARS-CoV (MOI = 0.1). The growth kinetics of SARS-CoV in culture supernatant and proportion of SARS-CoV-infected 2B4 cells were assessed at indicated time points by the standard Vero E6-based infectivity assay of the resulting cell-free supernatants (D) and infectious center assay (E). Finally, 2B4 cells (passage #6) were infected with SARS-CoV (MOI = 0.1) for 24, 48, and 72 hrs before being fixed with 4% paraformaldehyde for monitoring the morphological changes of infected cells, as visualized by the expression of SARS-CoV NP protein (red) by using the standard IHC (F).</p

Common up-regulated genes in 12, 24 and 48 hrs.

a<p>Enriched TF responsible for up-regulating the expression of genes at 12 hrs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008729#pone-0008729-g003" target="_blank">Fig. 3</a>).</p