The Molecular Mechanism Of Porcine Parvovirus Replication Is Suppressed By The ER Stress And The Nuclear Transport Mechanisms Of The Nonstructural Protein 1 Of Porcine Parvovirus

Endoplasmic reticulum(ER)stress and the unfolded protein response(UPR)are associated with numerous mammalian diseases,especially viral diseases.Porcine parvovirus(PPV)is a small single-stranded DNA virus and the causative agent of reproductive failure in swine.Although previous studies have shown that PPV infection activates ER stress,there is a need to better understand the specific molecular mechanisms of PPV-induced ER stress on virus replication.Here,we observed that the PPV infection of porcine kidney 15(PK-15)and porcine testis(PT)cells resulted in the activation of ER stress sensors mediated by protein kinase R-like ER kinase(PERK),but not inositol-requiring enzyme 1(IRE1)and activating transcription factor 6(ATF6).The key roles of ER stress activation on virus egress were confirmed by treatment of infected cells by specific ER stress inducers(thapsigargin,and tunicamycin),which obviously blocked PPV attachment and egress.Inhibition of the PERK pathway by small interfering RNA(siRNA)-mediated depletion of proteins such as PERK,eukaryotic initiation factor 2(eIF2a),and activating transcription factor 4(ATF4)significantly enhanced PPV replication.Moreover,we also observed that PPV-induced ER stress accelerates apoptotic cell death,which then suppresses PPV replication.Overexpression of pro-apoptotic factor C/EBP homologous protein(CHOP)significantly reduces PPV infection,whereas knockdown of CHOP expression obviously promotes PPV infection.In addition,accelerated apoptosis by specific apoptosis agonist(nutlin-3)significantly inhibited PPV replication.Together,the results indicate that PPV infection activates ER stress through the PERK signaling pathway and that ER stress then inhibits further PPV replication by promoting apoptosis.NS1,the major non-structural protein,is believed to play an important role in PPV replication.We for the first time showed in the present study that NS1 dynamically shuttle between nucleus and cytoplasm,despite their subcellular localization predominantly appear in nucleus.NS1 contains two nuclear export signal(NES)at amino acids 283-291(designated NES2)and 602-608(designated NES1).NES1 and NES2 were identified to be functional and transferable NES,and their nuclear export activity was blocked by the leptomycin B,suggesting that NS1 was exported out of the nuclei depending on the chromosome region maintenance 1 pathway.Moreover,truncation and deletion mutant analysis showed that NS1 contains a bipartite nuclear localization signal(NLS)at amino acids 256-274.Co-immunoprecipitation assays showed that NS1 interacts with importins a5 and a7.The nucleocytoplasmic shuttling of proteins is determined by a NES and NLS.Thus,kinetics of NS 1 and heterokaryon assay showed that NS 1 is a nucleocytoplasmic protein.