Bovine coronavirus nucleocapsid suppresses IFN-β production by inhibiting RIG-I-like receptors pathway in host cells

Abstract

The present study aimed to explore if bovine coronavirus nucleocapsid (BCoV N) impacts IFN-β production in the host cells and to reveal further molecular mechanism of BCoV pathogenesis. Human embryonic kidney (HEK) 293 T cells were transiently transfected with pMyc-BCoV-N recombinant plasmids, then infected with the vesicular stomatitis virus (VSV). Expression levels of beta interferon (IFN-β) mRNA were detected using RT-qPCR. The results showed that BCoV N gene was 1347 bp that was consistent with the expected size. pMyc-BCoV-N recombinant protein was 1347 bp which was successfully transcribed and overexpressed in HEK 293 T cells. BCoV-N recombinant protein inhibited dose-dependently VSV-induced IFN-β production (p < 0.01). MDA5, MAVS, TBK1 and IRF3 could promote transcription levels of IFN-β mRNA. But, BCoV-N protein demoted IFN-β transcription levels induced by MDA5, MAVS, TBK1 and IRF3. Furthermore, expression levels of MDA5, MAVS, TBK1 and IRF3 mRNAs were reduced in RIG-I-like receptor (RLR) pathway. In conclusion, BCoV-N reduced IFN-β levels in RIG-I-like receptor (RLR) pathway in HEK 293 T cells which were induced by MDA5, MAVS, TBK1 and IRF3(5D). BCoV-N protein inhibited IFN-β production and activation of RIG-I-like receptors (RLRs) signal pathway. Our findings demonstrated BCoV N protein is an IFN-β antagonist through inhibition of MDA5, MAVS, TBK1 and IRF3(5D) in RLRs pathway, also revealed a new mechanism of BCoV N protein to evade host innate immune response by inhibiting type I IFN production, which is beneficial to developing novel prevention strategy for BCoV disease in the animals and humans.

Keywords: Bovine coronavirus; IFN-β; Nucleocapsid protein; RIG-I-like receptor.

Fig. 1

BCoV-N PCR product and double enzyme digestion of pMyc-BCoV-N recombinant protein. a PCR product of BCoV-N, M: 1000 bp DNA Marker; 1: BCoV-N with 1347 bp. b Duble enzyme digestion of pMyc-BCoV-N recombinant protein; M: 1000 bp DNA Marker; 1: pMyc-BCoV-N recombinant protein; 2: pMyc-BCoV-N recombinant protein product of double enzyme digestion; 3: pCMV-Myc protein; 4: pCMV-Myc plasmid product of double enzyme digestion. NC: negative control

Fig. 2

BCoV N protein dose-dependently inhibited IFN-β production induced by VSV. HEK 293 T cells were cotransfected with vesicular stomatitis virus (VSV) and pMyc-BCoV-N. The outcomes indicated that BCoV N proteins depressed dose-dependently (500, 1000, 1500 or 2000 ng) levels of IFN-β mRNA. ***P < 0.001 as compared to VSV treatment

Fig. 3

Effect of BCoV N on transcriptional levels of VSV-mediated ISG15 and ISG56. BCoV N protein inhibited remarkably transcriptional levels of VSV-mediated ISG15 and ISG56. **P < 0.01 as compared to vector group (UT); ***P < 0.001 as compared to vector group (UT)

Fig. 4

Effects of BCoV N protein on the production of IFN-β induced by exogenous key factors. HEK 293 T cells were cotransfected with empty vector (pCMV-Myc), pMyc-BCoV-N protein or Flag-N-key factors (MDA5, MAVS, TBK1 and IRF3(5D)). a–d IFN-β expression levels induced by MDA5, MAVS, TBK1 and IRF3(5D), respectively. e Western blots of MDA5, MAVS, TBK1 and IRF3 (5D). These figure panels are taken from the different areas of the same gel or different gels and then joined together with a white space between the images. ***P < 0.001 as compared to pCMV-Myc group

Fig. 5

Effects of BCoV N protein on their self-transcription levels of four key proteins in signal pathway. ***P < 0.001 as compared to pCMV-Myc group