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
. 2012 Aug 6:9:149.
doi: 10.1186/1743-422X-9-149.

Non structural protein of avian influenza A (H11N1) virus is a weaker suppressor of immune responses but capable of inducing apoptosis in host cells

Sanjay Mukherjee  1 , Shamik Majumdar, Veena C Vipat, Akhilesh C Mishra, Alok K Chakrabarti
Affiliations

Non structural protein of avian influenza A (H11N1) virus is a weaker suppressor of immune responses but capable of inducing apoptosis in host cells

Sanjay Mukherjee et al. Virol J. .

Abstract

Background: The Non-Structural (NS1) protein of Influenza A viruses is an extensively studied multifunctional protein which is commonly considered as key viral component to fight against host immune responses. Even though there has been a lot of studies on the involvement of NS1 protein in host immune responses there are still ambiguities regarding its role in apoptosis in infected cells. Interactions of NS1 protein with host factors, role of NS1 protein in regulating cellular responses and apoptosis are quite complicated and further studies are still needed to understand it completely.

Results: NS1 genes of influenza A/Chicken/India/WBNIV2653/2008 (H5N1) and A/Aquatic bird/India/NIV-17095/2007(H11N1) were cloned and expressed in human embryonic kidney (293T) cells. Microarray based approach to study the host cellular responses to NS1 protein of the two influenza A viruses of different pathogenicity showed significant differences in the host gene expression profile. NS1 protein of H5N1 resulted in suppression of IFN-β mediated innate immune responses, leading to down-regulation of the components of JAK-STAT pathway like STAT1 which further suppressed the expression of pro-inflammatory cytokines like CXCL10 and CCL5. The degree of suppression of host immune genes was found considerable with NS1 protein of H11N1 but was not as prominent as with H5N1-NS1. TUNEL assay analyses were found to be positive in both the NS1 transfected cells indicating both H5N1 as well as H11N1 NS1 proteins were able to induce apoptosis in transfected cells.

Conclusions: We propose that NS1 protein of both H5N1 and H11N1 subtypes of influenza viruses are capable of influencing host immune responses and possess necessary functionality to support apoptosis in host cells. H11N1, a low pathogenic virus without any proven evidence to infect mammals, contains a highly potential NS1 gene which might contribute to greater virus virulence in different gene combinations.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Western blot analysis of NS1 protein expression in transfected cells. Equal amount of cellular proteins isolated (10 μg) from transfected and control cell extracts were separated by 12.5% SDS-polyacrylamide gel electrophoresis. Proteins transferred to Hybond-C membrane were probed with specific monoclonal antibodies against Influenza A-NS1. A known Influenza A H1N1 NS1 construct was used as a positive control.
Figure 2
Figure 2
A Venn-diagram representation of the number of host genes and the major pathways affected by NS1 protein of the two Influenza A viruses (H5N1 and H11N1) compared to mock. The number of genes exclusively affected by vector (mock) are shown in light green (5) were found to be mainly involved in stress response. Genes exclusively affected by the transfection of NS1 protein are represented in light red (16) for H5N1-NS1 and light blue (10) for H11N1-NS1; Gene ontology analysis showed that these were mainly involved in apoptosis, immune response and signal transduction processes. The differentially expressed genes common between vector and H11N1-NS1 are shown in dark blue (1) whereas, genes common between vector and H5N1-NS1 are shown in dark green (8). Three genes were common in transfections with all the different plasmid constructs. All analyses are in comparison with control untransfected cells.
Figure 3
Figure 3
Heat-map showing expression of apoptotic genes in 293 T cells transfected with pcDNA3.0 vector, H5N1-NS1 and H11N1-NS1 plasmid constructs. The expression was compared with control (untransfected) cells. The maps are representative of 3 replicates of microarray experiment.
Figure 4
Figure 4
Heat-map showing expression of immune genes in 293T cells transfected with pcDNA3.0 vector, H5N1-NS1 and H11N1-NS1 plasmid constructs. The expression was compared with control (untransfected) cells. The maps are representative of 3 replicates of microarray experiment.
Figure 5
Figure 5
Real-time PCR analysis of Immune and apoptotic genes in 293T cells. Total RNA isolated from control transfected cells as well as from cells transfected with pcDNA 3.0 (-) vector, H5N1-NS1 construct and H11N1-NS1 construct were used for real-time PCR analysis. The expression was analyzed compared to controls (untrasfected cells). Expression of β-Actin gene was used as an internal control. The data is representative of 3 replicates. Error bars indicate mean+/- standard deviation.
Figure 6
Figure 6
TUNEL assay analysis of Apoptosis in control and NS1 transfected cells. The cells were analyzed 24 h post transfection using fluorescently labeled anti-BrdU antibodies. The fluorescence was detected microscopically. Left panel are cells visualized under visible light without filter; Right panel are cells visualized under specific filters for fluorescence detection. The left and the right panel are the cells under same microscopic field A. Untransfected control cells B. Cells transfected with pcDNA 3.0 (-) vector C. cells transfected with pcDNA3-H5N1-NS1 construct D. cells transfected with pcDNA3-H11N1-NS1 construct.
Figure 7
Figure 7
A. Alignment of amino acid residues of NS1 protein of H5N1 and H11N1 influenza A viruses. Red circle indicates the truncation region of H5N1-NS1 protein sequence; red arrows indicate the positions of amino acid mismatches between the two sequences. B. 3D Models of NS1 proteins from H5N1 and H11N1 viruses. The differences are highlighted in red arrows.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Palese P, Shaw ML. In: Fields Virology. 5. Knipe DM, Howley PM, editor. Philadelphia: Lippincott Williams and Wilkins; 2008. Orthomyxoviridae: The viruses and their replication; pp. 1647–1689.
    1. Hale BG, Randall RE, Ortı’n J, Jackson D. The multifunctional NS1 protein of influenza A viruses. J Gen Virol. 2008;89:2359–2376. doi: 10.1099/vir.0.2008/004606-0. - DOI - PubMed
    1. Fukuyama S, Kawaoka Y. The pathogenesis of influenza virus infections: the contributions of virus and host factors. Curr Opin Immunol. 2011;23:481–486. doi: 10.1016/j.coi.2011.07.016. - DOI - PMC - PubMed
    1. Ehrhardt C, Seyer R, Hrincius ER, Eierhoff T, Wolff T, Ludwig S. Interplay between influenza A virus and the innate immune signaling. Microbes Infect. 2010;12:81–87. doi: 10.1016/j.micinf.2009.09.007. - DOI - PubMed
    1. Gack MU, Albrecht RA, Urano T, Inn KS, Huang IC, Carnero E, Farzan M, Inoue S, Jung JU, Garcia-Sastre A. Influenza A virus NS1 targets the ubiquitin ligase TRIM25 to evade recognition by the host viral RNA sensor RIG-1. Cell Host Microbe. 2009;5:439–449. doi: 10.1016/j.chom.2009.04.006. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

Associated data