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. 2020 Aug:247:108793.
doi: 10.1016/j.vetmic.2020.108793. Epub 2020 Jul 13.

Cellular poly(C) binding protein 2 interacts with porcine epidemic diarrhea virus papain-like protease 1 and supports viral replication

Pengfei Zhang  1 , Linyang Yu  1 , Jianguo Dong  2 , Yanling Liu  1 , Leyi Zhang  1 , Pengshuai Liang  1 , Lei Wang  1 , Bin Chen  2 , Li Huang  3 , Changxu Song  4
Affiliations

Cellular poly(C) binding protein 2 interacts with porcine epidemic diarrhea virus papain-like protease 1 and supports viral replication

Pengfei Zhang et al. Vet Microbiol. 2020 Aug.

Abstract

Porcine epidemic diarrhea virus (PEDV) belongs to the Alphacoronavirus genus in the Coronaviridae family. Similar to other coronaviruses, PEDV encodes two papain-like proteases. Papain-like protease (PLP)2 has been proposed to play a key role in antagonizing host innate immunity. However, the function of PLP1 remains unclear. In this study, we found that overexpression of PLP1 significantly promoted PEDV replication and inhibited production of interferon-β. Immunoprecipitation and mass spectrometry were used to identify cellular interaction partners of PLP1. Host cell poly(C) binding protein 2 (PCBP2) was determined to bind and interact with PLP1. Both endogenous and overexpressed PCBP2 co-localized with PLP1 in the cytoplasm. Overexpression of PLP1 upregulated expression of PCBP2. Furthermore, overexpression of PCBP2 promoted PEDV replication. Silencing of endogenous PCBP2 using small interfering RNAs attenuated PEDV replication. Taken together, these data demonstrated that PLP1 negatively regulated the production of type 1 interferon by interacting with PCBP2 and promoted PEDV replication.

Keywords: Coronavirus; Interaction; PEDV; Papain-like proteases; Poly(C) binding protein 2; Virus replication.

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Figures

Fig. 1
Fig. 1
Overexpression of PLP1 promotes PEDV replication and inhibits IFN-β promoter activation induced by TNF-α. (A) Vero E6 cells were transfected with pCMV-PLP1-HA or empty pCMV-HA vector. At 24 hpt, cells were infected with PEDV at MOI of 0.1. At 24 hpi, viral RNA levels were determined. (B) HEK293 T cells were seeded in a 24-well plate and cotransfected with IFN-Luc, TK-Luc, and pCMV-PLP1-HA or empty pCMV-HA vector. At 24 hpt, the cells were stimulated with 10 ng/mL TNF-α for 6 h and cell lysates were prepared for luciferase analysis. (C) HEK293 T were stimulated with TNF-α (10 ng/mL), then transfected with pCMV-PLP1-HA or empty pCMV-HA vector. Levels of IFN mRNA were determined by quantitative RT-PCR at 12, 24, 36 and 48 hpi.
Fig. 2
Fig. 2
Western blotting and silver staining of immunoprecipitated PLP1 samples. (A and B) HEK293 T cells were transfected with pCMV-PLP1-HA or empty pCMV-HA vector. At 24 hpt, the cells were lysed and the proteins were immunoprecipitated using the Pierce® HA Tag IP/Co-IP Kit. The immunoprecipitated proteins were analyzed by western blotting (A) and by silver staining (B).
Fig. 3
Fig. 3
GO and KEGG analysis of the PLP1 interactome. The LC–MS/MS results were analyzed by(A) GO and (B) KEGG pathway analysis.
Fig. 4
Fig. 4
Identification of proteins that interact with PLP1. HEK293 T cells were seeded in a six-well plate and cotransfected with pCMV-PLP1-HA, pECMV-3×FLAG-PCBP1, pECMV-3×FLAG-PCBP2 or empty vector. At 24 hpt, cells were lysed and immunoprecipitated with mouse anti-FLAG antibody. Immunoprecipitated samples were analyzed by western blotting.
Fig. 5
Fig. 5
Co-localization of PCBP2 with PLP1. (A) Vero E6 cells were cotransfected with pCMV-PLP1-HA and pECMV-3×FLAG-PCBP2 or empty pCMV-HA vector. The cells were assessed using an indirect immunofluorescence assay with anti-HA and anti-FLAG antibodies. (B) Co-localization of PLP1 and endogenous PCBP2 in Vero E6 cells. The cells were transfected with pCMV-PLPL-HA or empty pCMV-HA vector and then subjected to the IFA with anti-HA and anti-PCBP2 antibodies.
Fig. 6
Fig. 6
PLP1 up-regulates PCBP2 expression. Vero 6 cells were transfected with different concentrations of pCMV-PLP1-HA or empty pCMV-HA vector and western blotting was performed using anti-HA, anti-PCBP2 and anti-tubulin antibodies.
Fig. 7
Fig. 7
PCBP2 affects PEDV infection. IPEC-J2 cells were transfected with three different PCBP2-specific siRNAs. After 24 h, PCBP2 protein expression was assessed by western blotting with anti-PCBP2 and anti-tubulin antibodies. (B to C) IPEC-J2 cells were transfected with siRNA1, then 24 h later infected with PEDV for 24 h and 48 h. PCBP2 mRNA levels and PEDV RNA levels were determined by quantitative RT-PCR. (D) IPEC-J2 cells were transfected with pECMV-3×FLAG-PCBP2 or empty vector. At 24 hpt, the cells were infected with PEDV at a MOI of 0.1. PEDV viral loads were determined at 24, 48 and 60 hpi by quantitative RT-PCR.
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