Release of Severe Acute Respiratory Syndrome Coronavirus Nuclear Import Block Enhances Host Transcription in Human Lung Cells
Amy C. Sims<SUP>a</SUP>, Susan C. Tilton<SUP>d</SUP>, Vineet D. Menachery<SUP>a</SUP>, Lisa E. Gralinski<SUP>a</SUP>, Alexandra Sch?fer<SUP>a</SUP>, Melissa M. Matzke<SUP>d</SUP>, Bobbie-Jo M. Webb-Robertson<SUP>d</SUP>, Jean Chang<SUP>e</SUP>, Maria L. Luna<SUP>d</SUP>, Casey E. Long<SUP>a</SUP>, Anil K. Shukla<SUP>d</SUP>, Armand R. Bankhead III<SUP>f</SUP>, Susan E. Burkett<SUP>c</SUP>, Gregory Zornetzer<SUP>e</SUP>,*, Chien-Te Kent Tseng<SUP>g</SUP>, Thomas O. Metz<SUP>d</SUP>, Raymond Pickles<SUP>b</SUP>,<SUP>c</SUP>, Shannon McWeeney<SUP>f</SUP>, Richard D. Smith<SUP>d</SUP>, Michael G. Katze<SUP>e</SUP>,<SUP>h</SUP>, Katrina M. Waters<SUP>d</SUP> and Ralph S. Baric<SUP>a</SUP>,<SUP>b</SUP>
<SUP></SUP>
Author Affiliations: <SUP>a</SUP>Departments of Epidemiology <SUP>b</SUP>Microbiology and Immunology <SUP>c</SUP>Cystic Fibrosis Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA <SUP>d</SUP>Pacific Northwest National Laboratory, Richland, Washington, USA <SUP>e</SUP>Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington, USA <SUP>f</SUP>Oregon Health Sciences University, Portland, Oregon, USA <SUP>g</SUP>University of Texas Medical Branch, Department of Microbiology and Immunology, Galveston, Texas, USA <SUP>h</SUP>Washington National Primate Research Center, University of Washington, Seattle, Washington, USA
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.
FOOTNOTES
Received 17 October 2012. Accepted 17 January 2013.
Address correspondence to Amy C. Sims, sims0018@ad.unc.edu.
* Present address: Gregory Zornetzer, Institute for Systems Biology, Seattle, Washington, USA.
A.C.S. and S.C.T. contributed equally to this article.
Published ahead of print 30 January 2013
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JVI.02520-12.
Copyright ? 2013, American Society for Microbiology. All Rights Reserved.
-Amy C. Sims<SUP>a</SUP>, Susan C. Tilton<SUP>d</SUP>, Vineet D. Menachery<SUP>a</SUP>, Lisa E. Gralinski<SUP>a</SUP>, Alexandra Sch?fer<SUP>a</SUP>, Melissa M. Matzke<SUP>d</SUP>, Bobbie-Jo M. Webb-Robertson<SUP>d</SUP>, Jean Chang<SUP>e</SUP>, Maria L. Luna<SUP>d</SUP>, Casey E. Long<SUP>a</SUP>, Anil K. Shukla<SUP>d</SUP>, Armand R. Bankhead III<SUP>f</SUP>, Susan E. Burkett<SUP>c</SUP>, Gregory Zornetzer<SUP>e</SUP>,*, Chien-Te Kent Tseng<SUP>g</SUP>, Thomas O. Metz<SUP>d</SUP>, Raymond Pickles<SUP>b</SUP>,<SUP>c</SUP>, Shannon McWeeney<SUP>f</SUP>, Richard D. Smith<SUP>d</SUP>, Michael G. Katze<SUP>e</SUP>,<SUP>h</SUP>, Katrina M. Waters<SUP>d</SUP> and Ralph S. Baric<SUP>a</SUP>,<SUP>b</SUP>
<SUP></SUP>
Author Affiliations: <SUP>a</SUP>Departments of Epidemiology <SUP>b</SUP>Microbiology and Immunology <SUP>c</SUP>Cystic Fibrosis Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA <SUP>d</SUP>Pacific Northwest National Laboratory, Richland, Washington, USA <SUP>e</SUP>Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington, USA <SUP>f</SUP>Oregon Health Sciences University, Portland, Oregon, USA <SUP>g</SUP>University of Texas Medical Branch, Department of Microbiology and Immunology, Galveston, Texas, USA <SUP>h</SUP>Washington National Primate Research Center, University of Washington, Seattle, Washington, USA
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.
FOOTNOTES
Received 17 October 2012. Accepted 17 January 2013.
Address correspondence to Amy C. Sims, sims0018@ad.unc.edu.
* Present address: Gregory Zornetzer, Institute for Systems Biology, Seattle, Washington, USA.
A.C.S. and S.C.T. contributed equally to this article.
Published ahead of print 30 January 2013
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JVI.02520-12.
Copyright ? 2013, American Society for Microbiology. All Rights Reserved.
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