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. 2018 Jul 27;49(1):75.
doi: 10.1186/s13567-018-0571-5.

Annexin A2 binds to vimentin and contributes to porcine reproductive and respiratory syndrome virus multiplication

Xiao-Bo Chang  1 , Yong-Qian Yang  1 , Jia-Cong Gao  1 , Kuan Zhao  1 , Jin-Chao Guo  1 , Chao Ye  1 , Cheng-Gang Jiang  1 , Zhi-Jun Tian  1 , Xue-Hui Cai  1 , Guang-Zhi Tong  2 , Tong-Qing An  3
Affiliations

Annexin A2 binds to vimentin and contributes to porcine reproductive and respiratory syndrome virus multiplication

Xiao-Bo Chang et al. Vet Res. .

Erratum in

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important globally distributed and highly contagious pathogen that has restricted cell tropism in vivo and in vitro. In the present study, we found that annexin A2 (ANXA2) is upregulated expressed in porcine alveolar macrophages infected with PRRSV. Additionally, PRRSV replication was significantly suppressed after reducing ANXA2 expression in Marc-145 cells using siRNA. Bioinformatics analysis indicated that ANXA2 may be relevant to vimentin, a cellular cytoskeleton component that is thought to be involved in the infectivity and replication of PRRSV. Co-immunoprecipitation assays and confocal analysis confirmed that ANXA2 interacts with vimentin, with further experiments indicating that the B domain (109-174 aa) of ANXA2 contributes to this interaction. Importantly, neither ANXA2 nor vimentin alone could bind to PRRSV and only in the presence of ANXA2 could vimentin interact with the N protein of PRRSV. No binding to the GP2, GP3, GP5, nor M proteins of PRRSV was observed. In conclusion, ANXA2 can interact with vimentin and enhance PRRSV growth. This contributes to the regulation of PRRSV replication in infected cells and may have implications for the future antiviral strategies.

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Figures

Figure 1
Figure 1
ANXA2 was identified as a differentially upregulated protein by 2D-DIGE/MS analysis. A PAMs were infected with PRRSV HuN4 at a MOI of 0.01. At 48 hpi, cells were harvested and analyzed by 2D-DIGE. The 2D-DIGE profiles of differentially expressed protein levels in either the uninfected control PAM or HuN4-infected PAM. The 3D spot intensity represents the differentially expressed ANXA2 protein. B ANXA2 was identified using Western blotting. PAMs were infected with PRRSV HuN4 at a MOI of 0.01. At 48 hpi, cells were harvested for analysis by Western blotting. C The relative intensity ratios of ANXA2. D Bioinformatics analysis about ANXA2 was conducted using STRING and the interaction network revealed that ANXA2 and other proteins (including vimentin) form a protein interaction network.
Figure 2
Figure 2
ANXA2 knockdown by siRNA and its effect on PRRSV multiplication. A Reduced ANXA2 expression by siRNA. Marc-145 cells transfected with 100 nM siANXA2 or a non-targeting control (siNC), transfection reagent (mock treatment), or left untreated (no treatment; NT) for 36 h were infected with PRRSV HuN4 at a MOI of 0.01 for 24 and 48 h, and then collected the samples for Western blotting using mouse anti-β actin monoclonal antibody (GenScript), rabbit anti-ANXA2 monoclonal antibody (Cell Signaling Technology) and mouse anti-PRRSV N protein monoclonal antibody. B The relative intensity ratios of N protein and C ANXA2 in a Western blot showing successful knockdown. D PRRSV replication in ANXA2 knockdown cells obtained from cell culture supernatants treated with PRRSV HuN4 was assessed by RNA copy number by quantitative real-time RT-PCR assay and E PRRSV protein titers. *P < 0.05; **P < 0.01.
Figure 3
Figure 3
Co-localization of ANXA2 and vimentin in PAMs, Marc-145 cells. PAMs and Marc-145 cells at 70% confluence were washed, fixed, blocked with 5% skimmed milk and then incubated with rabbit anti-ANXA2 mAb (Cell Signaling Technology) or mouse anti-vimentin mAb (Cell Signaling Technology) at 4 °C overnight and then incubated for 1 h with goat anti-rabbit IgG-FITC antibodies or goat anti-mouse IgG-TRITC antibodies. Finally, the cells were then counter-stained with DAPI and subjected to confocal assay. Fluorescent staining of ANXA2 (green) and vimentin (red) in PAMs and Marc-145 cells, counterstained with DAPI (blue). The scale bar indicates 10 µm.
Figure 4
Figure 4
ANXA2 interacts with vimentin. PAMs (A) and Marc-145 cells (B) were harvested and subjected to co-IP assay using anti-ANXA2 monoclonal antibody and the precipitated proteins were analyzed by Western blotting using antibodies against ANXA2 and vimentin proteins.
Figure 5
Figure 5
Interaction between ANXA2-Flag and vimentin-HA in 293T cells. 293T cells co-transfected with pCMV-ANXA2-Flag and pCMV-vimentin-HA were harvested at 48 h and subjected to co-IP assay using mouse A anti-HA affinity gel or B anti-Flag affinity gel using. The precipitated proteins were analyzed by Western blotting using antibodies against Flag or HA.
Figure 6
Figure 6
Interaction between vimentin and a series of ANXA2-Flag mutants. A Schematic representation of the predicted protein domains of ANXA2. The full-length ANXA2 protein and five truncated ANXA2 mutants were examined in this study. B 293T cells co-transfected with pVIM-HA and plasmids expressing the ANXA2 mutants were collected at 48 h and subjected to co-IP assay using mouse anti-HA affinity gel. The precipitated proteins were analyzed by Western blotting using antibodies against Flag or HA.
Figure 7
Figure 7
ANXA2/vimentin binding to PRRSV structural proteins. A Validation of the reactivity of PRRSV structural protein mAbs and the PRRSV proteins by SDS-PAGE. B The mixture of PRRSV protein incubated with the extraction of HEK293T transfected with plasmids expressing tagged ANXA2 protein and subjected to Co-IP. The precipitated proteins were performed using anti-GP2, anti-GP3, anti-GP5, anti-M, and anti-N mAbs. C The binding of vimentin to PRRSV structural proteins. The mixture of PRRSV protein incubated with the extraction of HEK293T transfected with plasmids expressing tagged vimentin protein and then subjected to Co-IP. D The binding of the ANXA2-vimentin complex to PRRSV structural proteins. The mixture of PRRSV protein mixed with ANXA2-vimentin complex, and then performed the Co-IP assay. The triangle (black up-pointing triangle) highlights the position of the PRRSV N protein. E The mixture of PRRSV protein mixed with HEK293T transfected with plasmids expressing tagged ANXA2 and vimentin protein or control vector, and then performed Co-IP assay using anti-Flag affinity gel. The precipitated proteins were tested by Western blotting using the corresponding antibodies.
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