Molecular Mechanism Of Apoptosis Modulated By Highly Pathogenic PRRSV And Its Nonstructural Proteins

Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to share sophisticated pathogenesis. To explore the modulatory effect of PRRSV on host cell apoptosis will provide scientific foundation for better understanding its pathogenesis. In the present study, the apoptosis modulated by the Chinese highly pathogenic PRRSV (HP-PRRSV) and its molecular mechanism were analyzed, and meanwhile the roles and molecular basis of nonstructural proteins (Nsp) of PRRSV in the induced apoptosis were investigated. Moreover, the difference of apoptosis induced by PRRSV strains with different pathogenicity was compared.Western blot, flow cytometry and immunofluorescence assay (IFA) were employed in this study to analyze the apoptosis of HP-PRRSV-infected MARC-145 cells qualitatively and quantitatively. The results showed that the cleavage of PARP induced by straurosporine was significantly inhibited at 6 h and 8 h post-infection (pi) (P<0.01), indicating that PRRSV suppresses the apoptosis in early stage of infection. At 24 h pi, the release of cytochrome c from mitochondria, obvious activation of caspases-3 and cleavage of PARP were observed, suggesting that a significant pro-apoptotic activity appeared at the late stage of infection. Subsequent investigation on the activated apoptosis signaling pathways indicated that extrinsic pathway of apoptosis was induced by PRRSV infection at 16 h pi with apparent cleavage of procaspases-8. Whereas activated caspase-9 and caspase-12 could be detected at 24 h pi, suggesting that both mitochondria pathway and ER stress pathway could be induced by PRRSV infection. By means of detecting the caspase-3-or caspase-8-activated cells, the results further confirmed that caspase-3-and caspase-8-activated cells were only in virus-infected cells, no activation of caspases were observed in non-infected bystander cells. The cleavage of caspase-8 and PARP was shown in PAMs, indicating the resembling apoptosis induction in HP-PRRSV-infected-PAMs.In order to analyze the roles and molecular basis of apoptosis induced by the Nsps of PRRSV, western blot was used to examine the cleavage of PARP and the induction of apoptosis in cells that were expressing individual Nsp by using lentiviral packing system. Meanwhile, IFA was performed to detect the activation of caspsase-3 in Nsps-expressing cells by transient transfection. The results showed that caspase-3 could be activated significantly and obvious cleavage of PARP could be detected in MARC-145 cells that were expressing Nsp4 or Nsp10, suggesting these two proteins could induce apoptosis (P＜0.001). Further study verified that Nsp4 could activate caspase-8 and caspase-9/-12 to induce both extrinsic and intrinsic pathways. Meanwhile, Nsp4 was shown to activate the BH3-only protein Bim and degrade anti-apoptotic protein Bcl-xL, and Nsp10 could promote the cleavage of procaspases-8 and expression of Bid, and induce the truncation of Bid protein which is in the cytosol belonging to BH3-only family. The truncated tBid was shown to translocate to the outer mitochondrial membrane. Eventually, those alterations resulted in the mitochondrial outer membrane permeabilization and activation of the caspase-9 in the downstream causing the cleavage of PARP and apoptosis. Interferring of Bid expression caused less PARP cleavage triggered by Nspl0, however, inhibition of caspase-8 activity blocked apoptosis induced by Nspl0, indicating that Nspl0-induced apoptosis might be dependent on caspase-8 and Bid activation, and the activity of caspase-8 was essential for Nspl0-induced apoptosis. In contrast, Nsp3, Nsp5 and Nsp8 were able to block the staurosporine-induced PARP cleavage to suppress apoptosis. Molecular basis analysis suggested that Nsp3 was able to promote the phosphorylation of ser473 on Akt and serl36 of Bad, and increase the expression of anti-apoptotic protein Bcl-xL, which contributes to its inhibitory effect on apoptosis.To study the difference of the induced apoptosis between PRRSV strains with different pathogenicity, western blot and flow cytometry were used to analyze the apoptosis induced by HB-1/3.9 and JXwn06 in MARC-145 cells. Compared with HP-PRRSV JXwn06, HB-1/3.9 could induce the activation of caspase-9 with obvious cleavage at 36 h pi. From 24 h pi to 48 h pi, the amount of cells with activated caspase-3 in HB-1/3.9-infected cells was higher than JXwn06-infected cells, with significant difference (P＜0.001). At 24 h pi, the degradation of PARP in HB-1/3.9-infected cells was obvious, and the significant difference of the PARP cleavage between HB-1/3.9 and JXwn06 existed at 24 h pi (P＜0.001). The results indicated that, low pathogenic PRRSV HB-1/3.9 could induce significant apoptosis of MARC-145 cells at the late stage of infection. Consistent with the molecular basis exerted by the Nspl0 of JXwn06, the Nspl0 of HB-1/3.9 could induce apoptosis as well, whereas Nspl0 of HB-1/3.9 was able to promote the cleavage of procaspase-8 leading to the degradation of PARP in contrast with the Nspl0 of JXwn06. The amount of caspase-3-activated cells induced by the Rv-HJnl0 containing the replaced fragment of Nspl0 with the backbone of HB-1/3.9 was lower than the wild-type HB-1/3.9 at 24 h pi (P＜0.001). The amount of Rv-JHnlO-infected cells with apoptosis was higher than its wild-type JXwn 06 induced at 24 h pi (P＜0.001). It was indicated that the amount of Rv-JHn 10-activated cells was higher than Rv-HJnl0-infected cells, with significant difference at 36 h pi (P＜0.001). The above results suggested that the apoptosis induced by HB-1/3.9 was significant than HP-PRRSV JXwn06 did, and Nspl0 was the major protein contributing the difference of apoptosis.Taken together, our studies indicated that:(i) HP-PRRSV exhibited a double-phase modulation effect on the apoptosis, namely its inhibitory effect at the early stage of infection and promotion effect at the late stage of infection; (ii) multiple Nsps of PRRSV could be involved in the modulation of apoptosis, in which Nsp 4 and Nspl0 could induce apoptosis, while Nsp3, Nsp5 and Nsp8 could inhibited apoptosis; (iii) the difference of apoptotis induced by PRRSV strains with different pathogenicity was closely related to viral Nspl0. Our results provide scientific evidence for elucidating the pathogenesis of the Chinese HP-PRRSV, and add the roles of the Nsps of PRRSV in viral pathogenicity.