Mechanisms Of ER Stress And Apoptosis In Porcine Circovirus Type 2 Infected PK-15 Cells

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD). PCV2 is classified in the genus Circovirus within the family Circoviridae. The PCV2 genome is a single-stranded circular DNA molecule of approximately 1.7 kb which contains three major open reading frames (ORFs) coding for the replication protein (ORF1), capsid protein (ORF2), and a protein with suggested apoptotic activity (ORF3). PCV2 is the smallest virus known to infect mammals. The virus primarily targets the lymphoid tissues, leading to lymphoid depletion and subsequent immunosuppression in pigs. The disease is exacerbated by immunostimulation or concurrent infections with other pathogens. Although much progress has been made toward understanding the pathogenetic aspects of PCV2 infection, there are still some important questions unanswered hitherto with regard to the pathogenesis of PCV2 infection.Apoptotic cell death could be one of the mechanisms of lymphoid depletion during PCV2 infection. It remains unknown if apoptosis is the direct response of host cells to viral proteins such as ORF3 or capsid protein (Cap) or indirect (or even secondary) host cell responses following autophagy or unfolded protein response (UPR). UPR, autophagy and apoptosis have been found to occur in succession in host cells undergoing persistent stresses via a number of common signaling mechanisms shared by these different cellular responses in deciding the cell fate. The endoplasmic reticulum (ER) is a cellular membrane organelle that plays pivotal roles in virus replication and maturation. Viruses could hijack the host translation apparatus to produce viral proteins accumulated in the ER lumen or even utilize the ER as their replication sites, leading to ER stress as part of the pathogenesis of several diseases. To cope with the deleterious effects of virus-induced ER stress, cells activate critical signaling pathways, including the UPR and intrinsic apoptotic cascades, to exert complex effects on virus replication and pathogenesis. Mounting evidence indicates that a large number of viruses are capable of eliciting ER stress with the majority being RNA viruses. It remains unknown if single stranded DNA viruses, such as PCV2, are able to manipulate UPR during their infection. We speculate there is a possible link of UPR with apoptosis during PCV2 infection.In this study, we attempted to address:(1) Is UPR activated in PCV2-infected cells? (2) The effects of UPR on the viral replication? (3) Is UPR, once activated, linked with apoptosis?1. PCV2 infection is capable of triggering UPR via selective activation of the PERK/eIF2a/ATF4/CHOP pathwayWe examined the ER stress marker GRP78 and the three branches of UPR marker molecules PERK/eIF2a, ATF6, IREla/XBP1 splicing by using Western blotting and RT-PCR. PCV2 infection initiated UPR by inducing expression of GRP78, the master regulator of the UPR pathways, both in PK-15 and porcine alveolar macrophage cells. PERK pathway was selectively activated without concomitant activation of the IRE1/XBP1 or ATF6 pathway. We found that the pro-survival transcriptional factor ATF4 in PCV2-infected cells was induced probably via selective up-regulation by PERK/p-eIF2a. Both ATF4 and CHOP were induced at a later time point than PERK/eIF2a (36-48 hpi vs 24 hpi) when the viral capsid protein expression was significantly elevated and remained high. Therefore, we propose that persistent PCV2 infection could lead to selective activation of PERK and its downstream molecules via the PERK/eIF2a/ATF4/CHOP axis. By transient expression, we found that both replicase (Rep) and capsid (Cap) proteins of PCV2 could induce ER stress as shown by increased phosphorylation of PERK with subsequent activation the eIF2a/ATF4/CHOP axis. ORF3 protein did not induce UPR.2. PCV2 deploys PERK pathway and GRP78 for its enhanced replication in PK-15 cellsTo understand the role of UPR in PCV2 replication, we targeted key molecules in PERK pathway by using inhibitors or by RNA interference. Inhibition of PERK or eIF2a dephosphorylation significantly reduced viral replication, as revealed by reduced expression of Cap and lower virus titers (TCIDso:GSK2606414:4.58 vs 2.88, P<0.01; siPERK:4.53 vs 2.75, P<0.01; Salubrinal:4.49 vs 2.25, P<0.01). These results indicate that PCV2 may utilize the PERK pathway for its replication. Over-expression of GRP78 by using inducers or by extrachromosomal transient expression or treatment with tauroursodeoxycholic acid,a chemical chaperone that reduces ER stress, could enhance viral capsid expression and/or viral titers (TCIDso: pcDNA3-GRP78:4.03 vs 5.13, P<0.05; BIX:4.13 vs 5.12, P<0.05; thapsigargin:2.90 vs 3.67, P<0.05; TUDCA:4.20 vs 4.90, P<0.05), while GRP78 knockdown significantly reduced virus replication (TCIDso:siGRP78:4.29 vs 2.96, P<0.05), indicating that these chaperones, endogenous or exogenous, could help correct folding of viral proteins.3. PCV2 induces apoptosis by activating the PERK/CHOP/Bcl-2 pathwayER stress linked with apoptosis is considered as mechanisms of some viral infections, such as hepatitis B virus. It is still controversial with regard to PCV2-induced apoptosis via ORF3. Here we report that PCV2 could induce apoptosis through downregulation of anti-apoptotic protein Bcl-2 and Bcl-xL as well as increased caspase-3 cleavage in PK-15 cells. FACS and TUNEL assays also confirmed the apoptotic response. PCV2 also induced over-expression of the proapoptotic factor CHOP, which is seen as a link of UPR to apoptosis. Both tauroursodeoxycholic acid and 4-phenylbutyric acid, chemical chaperons, could reduce PCV2-induced apoptosis by reducing CHOP and capase-3 cleavage. PCV2 infection can increase cytosolic Ca2+ via IP3R, a major calcium release channel on the sarco/endoplasmic reticulum (SR/ER). To further understand the role of UPR in PCV2-induced apoptosis, we inhibited PERK and IP3R. Both treatments reduced PCV2-induced apoptosis. Silencing of CHOP partically restored PCV2-induced downregulation of Bcl-2, indicating that CHOP inhibited Bcl-2 expression during infection. We sought to determine whether the expression of PCV2 Rep or Cap protein alone is capable of inducing apoptosis. By transient expression, we found that Cap expression, but not Rep, significantly reduced anti-apoptotic Bcl-2 and increased caspase-3 cleavage, possibly due to increased expression of CHOP. Since knockdown of PERK by RNA interference clearly reduced Cap-induced CHOP expression as well as less degree of caspase-3 cleavage and apoptotic cell death, possibly by partially rescuing Bcl-2 expression, we propose that there is connection between Cap-induced UPR and apoptosis via the PERK/eIF2α/ATF4/CHOP pathway.In conclusion, this study demonstrated that PCV2 elicits UPR and triggers apoptosis through induction of PERK/eIF2 α/ATF4/CHOP/Bcl-2 pathway and ER calcium depletion. Capsid protein plays a critical role in this process. PCV2 could utilize PERK and GRP78 to promote its replication, indicating that PCV2 regulates the pro-survival UPR in favor of its replication during early infection. Our findings provide new elements to understand the mechanisms of the pathogenesis of PCV2 infection and furnish insight in to development of antiviral drugs.