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

Abstract

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.

Cell Host Response to Infection with Novel Human Coronavirus EMC Predicts Potential Antivirals and Important Differences with SARS Coronavirus

“…1). While SARS-CoV has been previously shown to maintain steady replication and cell viability to 72 hpi (19), HCoV-EMC induced substantial cytopathic effect at 18 to 24 hpi, with significant cell rounding and detaching. Together, these data suggest that although HCoV-EMC and SARS-CoV exhibit similar replication kinetics, they elicit different host responses in lung epithelial cells.…”

“…The corresponding cells were harvested for transcriptomic analysis. SARS-CoV titers after infection of Calu-3 2B4 cells at an MOI of 5 were determined using the same method (19). The error bars represent the standard deviations among triplicate cell samples.…”

Diversification of importin-α isoforms in cellular trafficking and disease states

“…Although importin α1 as such does not import STAT1, it has been hypothesized that loss of α1 increases competition for importin-α5 binding, thereby reducing the net influx of STAT1 into the nucleus. A follow-up paper from the same group identified additional transcription factors, the transcription of which was disrupted by the ORF6 sequestration of importin α1, and some of these factors, such as cAMP response element-binding protein 1 (CREB1), SMAD4, p53 and Oct3/4, are important in the response to viral infection [96]. In addition to targeting importin α, the non-classic NLS of STAT1 is itself a target for reducing the antiviral response.…”

Epigenetic Landscape during Coronavirus Infection

“…SARS-CoV encodes several proteins that modulate innate immune signaling through the antagonism of the induction of interferon. As mentioned above, several nsps that are encoded by ORF1 (ORF1a/b), like nsp1, nsp3 papain-like protease, nsp14 and nsp16 antagonize various sensing or signaling programs and NFκβ, or function to cap viral messenger RNAs (mRNAs) and evade interferon-induced protein with tetratricopeptide repeats (IFIT) 1-3 ISGs [11,21,22,23,24,25] (Figure 1). These nsps show high homology to proteins of other human coronaviruses and are critical for efficient viral replication.…”

“…These nsps show high homology to proteins of other human coronaviruses and are critical for efficient viral replication. Several downstream open reading frames like ORF3a, ORF3b and ORF6 also antagonize sensing or signaling pathways, or block karyopherin 2 nuclear import [13,21] (Figure 1). MERS-CoV also encodes several luxury functions with interferon antagonism activities, including ORF4a, ORF4b and perhaps ORF5, noting that ORF4b antagonizes phosphodiesterase activity and RNAse L activation [26,27,28,29,30].…”