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, 17 (1), 191

Annexin A2 (ANXA2) Interacts With Nonstructural Protein 1 and Promotes the Replication of Highly Pathogenic H5N1 Avian Influenza Virus

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Annexin A2 (ANXA2) Interacts With Nonstructural Protein 1 and Promotes the Replication of Highly Pathogenic H5N1 Avian Influenza Virus

Yong Ma et al. BMC Microbiol.

Abstract

Background: Non-structural protein 1 (NS1) is a multifunctional protein and a crucial regulatory factor in the replication and pathogenesis of avian influenza virus (AIV). Studies have shown that NS1 can interact with a variety of host proteins to modulate the viral life cycle. We previously generated a monoclonal antibody against NS1 protein; In the current research study, using this antibody, we immunoprecipitated host proteins that interact with NS1 to better understand the roles played by NS1 in communications between virus and host.

Results: Co-immunoprecipitation experiments identified annexin A2 (ANXA2) as a target molecule interacting with NS1. Results from confocal laser scanning microscopy indicated that NS1 co-localized with ANXA2 in the cell cytoplasm. Overexpression of ANXA2 significantly increased the titer of H5N1 subtype HPAIV, whereas siRNA-mediated knockdown of ANXA2 markedly inhibited the expression of viral proteins and reduced the progeny virus titer.

Conclusions: Our results indicate that ANXA2 interacts with NS1 and ANXA2 expression increases HPAIV replication.

Keywords: ANXA2; H5N1 subtype HPAIV; NS1; Viral replication.

Conflict of interest statement

Ethics approval and consent to participate

Per the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural. Sciences’ Ethical Guidelines & Regulations, no formal ethics approval was required for this study.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
ANXA2 was confirmed as a novel host protein that binds to NS1. NS1-associated proteins from infected (InfA) or uninfected (Mock) A549 cell lysates were immunoprecipitated using the anti-NS1 D7 antibody, separated by SDS-PAGE (8%), and visualized by silver staining. The InfA group-specific band (indicated by an asterisk) was identified as ANXA2 by LTQ-MS
Fig. 2
Fig. 2
Validation of the interaction between ANXA2 and NS1. a Co-IP assay confirming the interaction between NS1 and ANXA2. A549 cells were transfected with HA-ANXA2 plasmid and infected with GD1322 24 h later. Cell lysates, harvested 36–48 h after infection, were subjected to IP and western blotting with anti-HA or anti-NS1 D7 antibodies. b Co-IP assay detecting the association between NS1 and endogenous ANXA2. A549 cells were infected with GD1322 and collected after 12 h. Next, cell lysates from infected or uninfected A549 cells were immunoprecipitated with an anti-ANXA2 rabbit antibody or the anti-NS1 D7 mouse antibody. After incubation with protein A/G-agarose beads, the Immunoprecipitation pellets were immunoblotted with the anti-NS1 D7 mouse antibody or the anti-ANXA2 rabbit antibody. c Pull down assay to verify that the ED of NS1 binds with ANXA2. Full-length NS1 and each functional domain were fused with GST and then incubated with uninfected A549 lysate. GST and ANXA2 were detected by western blotting. d Colocalization of ANXA2 and NS1 in A549 cells. HA-ANXA2 and FLAG-NS1 plasmids were co-transfected into A549 or 293 T cells. After incubation for 24 h, the cells were double-immunostained for FLAG-NS1 (green) and HA-ANXA2 (red). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue)
Fig. 3
Fig. 3
ANXA2 influences AIV replication in A549 cells. a Overexpression of ANXA2 in A549 cells. A549 cells were transfected with HA-ANXA2 plasmid or an empty vector (Vec) and then infected with GD1322 (MOI = 0.1). After 24 h, HA-ANXA2 and NS1 proteins were detected by western blotting. b Progeny virus titers increased significantly in A549 cells overexpressing ANXA2. Viral supernatants were collected at 12 h and 24 h after infection. Viral titers were assayed based on hemagglutination, and the results are expressed as TCID50 per mL. c Knockdown efficiency of ANXA2. A549 cells were transfected with siNC or siANXA2. The results shown are from qPCR and western blotting analyses performed 24 h after infection. d Progeny virus titers decreased significantly after transfection with siANXA2 at 24 hpi. The results are presented as the mean and SD from three independent experiments. *, p<0.05 compared with cells transfected with empty vector or siNC. **, p<0.01 compared with cells transfected with empty vector or siNC
Fig. 4
Fig. 4
ANXA2 influences the replication of H5 subtype AIV. A549 cells were infected with GD1322 (MOI = 0.1) after transfection with siNC or siANXA2 for 24 h. The cells were then collected at 4, 12, 24 and 48 hpi to extract RNA or protein (a, b). A549 cells were also collected at 0, 4, 12, 24, and 48 hpi to extract protein (c). a qRT-PCR detection of gene synthesis of viral genes after transfection with siRNA. Total RNA was extracted from A549 cells, and ANXA2 mRNA, M vRNA and HA vRNA were used for relative quantification. GAPDH was used as a control. b Western blotting detection of viral protein expression after transfection of siRNA. After extracting total proteins from A549 cells, viral protein expression was determined using H5-specific antiserum from our lab. ANXA2 and tubulin were detected using commercial MAbs. The results are presented as the mean and SD from three independent experiments. c Western blotting detection of ANXA2 and HA expression after infection with H5N1 AIV. The same antibodies described above were used

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