Molecular Mechanism Of Interaction Of Porcine Reproductive And Respiratory Syndrome Virus Nsp4 With Cytochrome C1 And Its Induction On Apoptosis

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious pathogen which severely damages the swine industry worldwide. The emergence and prevalence of highly pathogenic PRRSV (HP-PRRSV) in swine industry have led to huge economic losses. PRRSV encodes 14 non-structural proteins (Nsp),in which Nsp4, one protein with 3C-like serine protease activity, plays an important role in virus replication, inhibition of host innate immune responses and apoptosis induction of host cells. In the present study, the Porcine alveolar macrophages (PAMs) protein Cytochrome c1 (Cyto.cl) that was a novel interactor of nonstructural protein Nsp4 of PRRSV was identified. Furthermore, the interaction between Nsp4 and Cyto.cl was confirmed and the molecular mechanism that PRRSV Nsp4 protein is involved in cell apoptosis was elucidated. Thus, the new biological function of Nsp4 was characterized to provide new clues for further study on PRRSV molecular pathogenesis.On the other hand, the lentiviral vector technology was used to generate the recombinant lentiviruses that contain Nsp4 gene from the representative HP-PRRSV strain (JXwn06) and mutant Nsp4 with inactive enzyme site. The recombinant lentiviruses were rescued and successfully transduced to MARC-145 cells.Although the cell lines that express the fusion protein were obtained, the cell lines that were stably expressing Nsp4 fusion protein was failed to obtain, maybe due to the characteristic of the apoptosis induction of Nsp4.To further explore the molecular mechanism that Nsp4 induces apoptosis, yeast two-hybrid assay with Nsp4 as the bait to screen the PAMs cDNA library was performed and the Cyto.cl protein that associated with apoptosis regulation or apoptosis signaling transduction was identified. The interaction between Nsp4 and Cyto.cl was further validated by using yeast two-hybrid system. The interaction domain of Nsp4 and Cyto.c1 was identified by Co-immunoprecipitation (Co-IP) and the confocal microscopy analysis. Our results showed that the N-terminus (1?160aa) of Nsp4 interacted with the N-terminus (1?230aa) of Cyto.cl.The siRNA was used to silence the endogenous Cyto.cl in the MARC-145 cells, and further analyse the effect of interaction between Nsp4 and Cyto.c1 on the apoptosis induced by Nsp4. The data showed that knockdown of Cyto.cl significantly inhibited the Nsp4-induced apoptosis. We also found that Nsp4 could cleave Cyto.cl both in PRRSV-infected and plasmid-transfected cells. The cleavage capacity was determined by its 3C-like serine protease activity in a dose-dependent manner.Furthermore, we found that Nsp4 specifically targeted Cyto.cl at the site of E230?G231. The truncated Cyto.cl (1?230aa) generated by the Cyto.cl cleavage with Nsp4 could obviously activate Caspase-3 and cause the destruction of mitochondria. Then mitochondria damage was produced, and mitochondria-mediated apoptosis was initiated. Finally apoptosis was induced.In summary, we identified that PRRSV Nsp4 could interact with the host cellular protein Cyto.c1 and uncovered the molecular mechanism of the interaction in Nsp4-induced host cell apoptosis. These findings will provide a valuable scientific basis to elucidate the pathogenesis of PRRSV.