Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Oct 12;37(2):109803.
doi: 10.1016/j.celrep.2021.109803.

Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription

Jingjing Pei  1 , Nina R Beri  2 , Angela J Zou  1 , Philipp Hubel  3 , Hannah K Dorando  2 , Valter Bergant  4 , Rebecca D Andrews  2 , Jiehong Pan  5 , Jared M Andrews  2 , Kathleen C F Sheehan  2 , Andreas Pichlmair  6 , Gaya K Amarasinghe  2 , Steven L Brody  5 , Jacqueline E Payton  7 , Daisy W Leung  8
Affiliations

Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription

Jingjing Pei et al. Cell Rep. .

Abstract

Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.

Keywords: NS1; epigenetics; host gene transcription; immune antagonism; non-structural protein; respiratory syncytial virus.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. NS1 partitions into the nucleus in RSV-infected airway epithelial cells and associates with host nuclear proteins
hTECs differentiated on supported membranes using air-liquid interface culture were treated with PBS (mock) or RSV at MOI of 1 on the apical surface for 72 h. Fixed cells on membranes were processed through paraffin embedding and then immunostained using antibodies to RSV NS1 (green; left and right panels) or nucleoprotein (green; middle panel) and co-stained with cilia marker anti-acetylated tubulin (ac-α-tubulin, red). (A and B) Shown are representative photomicrographs from three experiments with (A) low-power observation and (B) high-power magnification. Scale bar: 10 μm. Merged images in the bottom rows of (A) and (B) demonstrate that NS1 localizes in cytoplasm and nucleus of cells, while nucleoprotein is present in aggregates within the cytoplasm of ciliated cells during RSV infection. (C) AP-LC-MS/MS reveals cytoplasmic and nuclear interactors of RSV NS1. Volcano plot shows p values and fold enrichment over unrelated control (n = 3). Significant interactors are depicted in red, other proteins in blue (non-significant) or gray (depleted).
Figure 2.
Figure 2.. Nucleocytoplasmic partitioning of NS1 is facilitated through interactions with host transporters
(A) Representative confocal micrographs of A549 cells that were mock infected (top rows) or infected with RSV A2 at a MOI of 1 for 24 h (bottom rows). Cells were fixed and stained with anti-NS1 antibody (green), anti-Mediator 1 (MED1) (red), or DAPI (blue). Scale bar: 10 μm. Right panels: intensity profile analysis was performed along the white line using ZEN 2.6 software to display the distribution of the three fluorophore signals. (B) Representative confocal images of A549 cells that were infected with RSV A2 at a MOI of 1 for 24 h and incubated with DMSO (control, left panel) or KPT-335 (1 μM, middle panel). Cells were fixed and stained with anti-exportin 1 (XPO1) (red), anti-NS1 antibody (green), or DAPI (blue). Scale bar: 10 μm. Cells were analyzed using ImageJ to determine the NS1 fluorophore signal of nucleus relative to cytoplasm (Fn / Fc, right part). Data represent mean ± SD. Statistical analysis was performed using Student’s t test (****p < 0.0001). (C) Representative confocal micrographs of A549 cells that were transfected either with GFP-KEAP1 (top rows) or GFP-NS1 (bottom rows) for 24 h. Cells were fixed and stained with anti-MED1 antibody (red) or DAPI (blue) and visualized for GFP fluorescence (green). Scale bar: 10 μm. Right panels: intensity profile analysis was performed along the white line using ZEN 2.6 software to display the distribution of the three fluorophore signals. (D) PCC was determined between MED1 and NS1 or MED1 and KEAP1 channels for GFP-NS1 and GFP-KEAP1 transfected cells as shown in (C). Representative of 3 experiments. (****p < 0.0001; ns, not significant)
Figure 3.
Figure 3.. NS1 binds at transcriptional regulatory elements of genes differentially expressed during RSV infection
(A) Western blots show co-IP of FLAG-NS1 with MED1, MED14, and MED25. WCL, whole-cell lysate; Cy, cytoplasmic fraction; SN, soluble nuclear fraction; Ch, chromatin fraction. (B) Western blots of subcellular fractionated A549 cells expressing HA-NS1. U, unfractionated; M, membrane; Cy, cytosol. (C) Overlap of NS1 and Mediator ChIP-seq peaks in A549 cells. (D) Bar chart shows number of NS1 ChIP-seq peaks in functional genomic regions. (E) Heatmap shows differentially expressed genes with 10 kb of NS1 peaks at 24, 48, and 96 hpi with RSV (RNA-seq). (F) NS1 ChIP-seq peaks coincide with an enhancer (A), promoter (C), and Mediator peaks (B–D). Tracks: ChIP-seq as reads per million (RPM). Red and blue bars: called peaks (HOMER); GeneHancer track shows overlapping promoter (pink) and enhancers (green) (UCSC browser). (G) Bar graph shows fold change + standard deviation of luciferase activity of reporters with NS1-bound regions in the IFIT locus (A–D as in (F) relative to minimal promoter reporter (minP) in A549 cells transfected with empty vector (EV) or NS1 and treated with LMW polyI:C for 24 h. Unpaired t test, *p < 0.05, **p < 0.005, ns, not significant. All are representative of ≥2 experiments.
Figure 4.
Figure 4.. The C-terminal helix of NS1 is important for modulating gene transcription
(A) Heatmap shows DEG within 10 kb of NS1 peaks at 96 hpi with mock, NS1 WT, or Y125A RSV (RNA-seq). (B and C) Volcano plots show (B) WT NS1 or (C) NS1 Y125A versus mock infection (as in A). Red: DEG <10 kb from an NS1 peak; blue: DEG >10 kb from an NS1 peak; gray: non-DEG. Selected genes are labeled. (D) Two transcription factor motifs enriched in NS1 peaks. The ISRE motif, which is highly similar to IRF1 and STAT1/STAT2 motifs, drives transcription of the luciferase reporter in (E). (A)–(D) are representative of 2 experiments. (E) Bar graph shows fold change + standard deviation of ISRE reporter luciferase activity relative to minimal promoter reporter (minP) in A549 cells transfected with empty vector (EV), WT, or Y125A NS1 and treated with LMW polyI:C for 24 h. Unpaired t test; **p < 0.005. Representative of >3 experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aherne W, Bird T, Court SD, Gardner PS, and McQuillin J (1970). Pathological changes in virus infections of the lower respiratory tract in children. J. Clin. Pathol. 23, 7–18. - PMC - PubMed
    1. Andersson R, Gebhard C, Miguel-Escalada I, Hoof I, Bornholdt J, Boyd M, Chen Y, Zhao X, Schmidl C, Suzuki T, et al. (2014). An atlas of active enhancers across human cell types and tissues. Nature 507, 455–461. - PMC - PubMed
    1. Andrews JM, El-Alawi M, and Payton JE (2018). Genotify: Fast, lightweight gene lookup and summarization. J. Open Source Softw. 3, 885. - PMC - PubMed
    1. Atreya PL, Peeples ME, and Collins PL (1998). The NS1 protein of human respiratory syncytial virus is a potent inhibitor of minigenome transcription and RNA replication. J. Virol. 72, 1452–1461. - PMC - PubMed
    1. Ban J, Lee NR, Lee NJ, Lee JK, Quan FS, and Inn KS (2018). Human Respiratory Syncytial Virus NS 1 Targets TRIM25 to Suppress RIG-I Ubiquitination and Subsequent RIG-I-Mediated Antiviral Signaling. Viruses 10, 716. - PMC - PubMed

Publication types

MeSH terms