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. 2017 Jul 11;14(1):125.
doi: 10.1186/s12985-017-0794-5.

Interleukin-2 enhancer binding factor 2 interacts with the nsp9 or nsp2 of porcine reproductive and respiratory syndrome virus and exerts negatively regulatory effect on the viral replication

Xuexia Wen  1 , Ting Bian  1 , Zhibang Zhang  1 , Lei Zhou  1 , Xinna Ge  1 , Jun Han  1 , Xin Guo  1 , Hanchun Yang  2 , Kangzhen Yu  3   4
Affiliations

Interleukin-2 enhancer binding factor 2 interacts with the nsp9 or nsp2 of porcine reproductive and respiratory syndrome virus and exerts negatively regulatory effect on the viral replication

Xuexia Wen et al. Virol J. .

Abstract

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failures in sows and respiratory diseases in growing pigs, resulting in huge economic loss for the pig production worldwide. The nonstructural protein 9 (nsp9) and nonstructural protein 2 (nsp2) of PRRSV are known to play important roles in viral replication. Cellular interleukin-2 enhancer binding factor 2 (ILF2) participates in many cellular pathways and involves in life cycle of some viruses. In the present study, we analyzed the interaction of cellular ILF2 with the nsp9 and nsp2 of PRRSV in vitro and explored the effect of ILF2 on viral replication.

Methods: The interaction of ILF2 with the nsp9 or nsp2 of PRRSV was analyzed in 293FT cells and MARC-145 cells by co-immunoprecipitation (Co-IP) and the co-localization of ILF2 with the nsp9 or nsp2 of PRRSV in MARC-145 cell and pulmonary alveolar macrophages (PAMs) was examined by confocal immunofluorescence assay. The effect of ILF2 knockdown and over-expression on PRRSV replication was explored in MARC-145 cells by small interfering RNA (siRNA) and lentivirus transduction, respectively.

Results: The interaction of ILF2 with nsp9 or nsp2 was first demonstrated in 293FT cells co-transfected with ILF2-expressing plasmid and nsp9-expressing plasmid or nsp2-expressing plasmid. The interaction of endogenous ILF2 with the nsp9 or nsp2 of PRRSV was further confirmed in MARC-145 cells transduced with GFP-nsp9-expressing lentiviruses or infected with PRRSV JXwn06. The RdRp domain of nsp9 was shown to be responsible for its interaction with ILF2, while three truncated nsp2 were shown to interact with ILF2. Moreover, we observed that ILF2 partly translocated from the nucleus to the cytoplasm and co-localized with nsp9 and nsp2 in PRRSV-infected MARC-145 cells and PAMs. Finally, our analysis indicated that knockdown of ILF2 favored the replication of PRRSV, while over-expression of ILF2 impaired the viral replication in MARC-145 cells.

Conclusion: Our findings are the first to confirm that the porcine ILF2 interacts with the nsp9 and nsp2 of PRRSV in vitro, and exerts negatively regulatory effect on the replication of PRRSV. Our present study provides more evidence for understanding the roles of the interactions between cellular proteins and viral proteins in the replication of PRRSV.

Keywords: Interaction; Interleukin-2 enhancer binding factor 2 (ILF2); Nonstructural protein 2 (nsp2); Nonstructural protein 9 (nsp9); Porcine reproductive and respiratory syndrome virus (PRRSV); Replication.

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Competing interests

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Analysis of cellular proteins interacting with PRRSV nsp9 by IP combined with LC-MS/MS assay. 3D4/21 cells were transduced with GFP-nsp9- or GFP-expressing lentiviruses (a) and then the expression of GFP-nsp9 and GFP in 3D4/21 cells were examined by western blotting (b). The cell lysates were immunoprecipitated with anti-GFP mAb, and were then separated by SDS-PAGE, and followed by silver staining (c). Shown are the differential protein bands between GFP-nsp9- and GFP-expressing 3D4/21 cells. Asterisk indicatess the band from which ILF2 was identified
Fig. 2
Fig. 2
Interaction of the nsp9 with ILF2. a and b The interaction of nsp9 with exogenous ILF2. 293FT cells were co-transfected with the indicated plasmids. The cell lysates were immunoprecipitated with an anti-HA mAb and detected with an anti-HA mAb or anti-Myc polyclonal antibody. c The interaction of nsp9 with endogenous ILF2. MARC-145 cells were transduced with GFP-nsp9- or GFP-expressing lentiviruses. The cell lysates were immunoprecipitated with an anti-GFP mAb and detected with an anti-GFP or anti-ILF2 mAb
Fig. 3
Fig. 3
Interaction of the nsp2 with ILF2. a and b The interaction of nsp2 with exogenous ILF2. 293FT cells were co-transfected with the indicated plasmids. The cell lysates were immunoprecipitated with an anti-HA mAb and further probed with an anti-HA mAb or anti-Myc polyclonal antibody. c The interaction of nsp2 with endogenous ILF2 and ILF3. MARC-145 cells were infected with PRRSV JXwn06 and then cell lysates were immunoprecipitated with an anti-nsp2 mAb, and were then detected with an anti-nsp2 or anti-ILF2 mAb or anti-ILF3 polyclonal antibody
Fig. 4
Fig. 4
The regions responsible for the interaction of ILF2 with nsp9 and nsp2. a The schematic diagram of the truncated nsp9—nsp9N (aa1-385) and nsp9C (aa386-640). b The interaction of ILF2 with nsp9C by Co-IP. 293FT cells were co-transfected with the indicated plasmids. The cell lysates were immunoprecipitated with an anti-HA mAb and further probed with an anti-HA or anti-Myc MAb. c The schematic diagram of three truncated nsp2—nsp2N (aa1-405), nsp2M (aa323-814) and nsp2C (aa323-1166). d The interaction of ILF2 with nsp2N, nsp2M and nsp2C by Co-IP. 293FT cells were co-transfected with the indicated plasmids. The cell lysates were immunoprecipitated with an anti-GFP mAb and further probed with an anti-GFP mAb or anti-Myc polyclonal antibody
Fig. 5
Fig. 5
Co-localization of ILF2 with nsp9 and nsp2 in MARC-145 cells (a) and PAMs (b). The cells were infected with PRRSV JXwn06 at a MOI of 0.01, and were then fixed and double-stained at 36 h post-infection with a rabbit anti-ILF2 polyclonal antibody and a mouse anti-nsp9 or anti-nsp2 mAb, and followed by the FITC-conjugated goat anti-rabbit IgG (green) and TRITC-conjugated goat anti-mouse IgG (red). Nuclei were stained with DAPI
Fig. 6
Fig. 6
Enhancement of PRRSV replication by ILF2 knockdown in MARC-145 cells. a MARC-145 cells were transfected with different concentrations of siRNAs targeting ILF2 (SiILF2) and the silencing efficiency of ILF2 was examined by western blotting with an anti-ILF2 mAb. The quantity of β-actin was used for normalization of the amount of ILF2 expression. The optical density ratios of ILF2/β-actin are shown with graphs. MARC-145 cells transfected with SiILF2 or negative control siRNA (SiNC), and normal MARC-145 cells, were infected with PRRSV JXwn06 at a MOI of 0.01. The silencing efficiency (b) and the virus titers were examined (c) at the indicated time points post-infection. Data are expressed as means ± SD of three independent experiments (*p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significant)
Fig. 7
Fig. 7
Inhibition of PRRSV replication by ILF2 over-expression in lentiviruses- transduced MARC-145 cells. Lentiviruses that were expressing GFP-ILF2 or GFP were transduced into MARC-145 cells (a) and then the expression of GFP-nsp9 and GFP in MARC-145 cells were examined by western blotting (b). The β-actin was used for normalization of the expression of ILF2. The transduced MARC-145 cells were infected with PRRSV JXwn06 at a MOI of 0.01. Normal MARC-145 cells were infected with PRRSV JXwn06 as a control. The virus yields were assayed at the indicated time points post-infection (c). Data are expressed as means ± SD of three independent experiments (*p < 0.05; ** p < 0.01; ns, no significant)
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