title: C-terminal region of apoptin affects chicken anemia virus replication and virulence.
abstract: BACKGROUND
  Chicken anemia virus (CAV) causes anemia and immune suppression, which are important diseases in the poultry industry. CAV VP3, also referred as 'apoptin', has been shown to selectively kill tumor cells, raising great hopes for its utilization as an anticancer therapy. The ability of apoptin to induce apoptosis is closely related to its nuclear localization. The C-terminal region of apoptin contains a bipartite nuclear localization signals (NLS), and a nuclear export signal (NES) is located between the arms of the NLS. Most previous studies have expressed apoptin of different lengths in vitro to understand the relationship between its localization and its induction of apoptosis.
  
  
  METHODS
  In this study, we investigated the replication of CAV and its induction of apoptosis in vitro and in vivo with VP3-truncated infectious virus. Quantitative PCR was used to detect viral replication in MDCC-MSB1 cells, and the viral localization was observed by confocal microscopy. Flow cytometry was uesed to analyze virus-induced apoptosis in MDCC-MSB1 cells. Additionally, chickens infected with the rescued viruses compared with the parental virus rM9905 to evaluate the viral replication in vivo and virulence.
  
  
  RESULTS
  Based on the infectious clone, we rescued two viruses in which were deleted NES-NLS2 (rCAV-VP3N88) or NLS1-NES-NLS2 (rCAV-VP3N80) in the C-terminal region of apoptin. The viral load of rCAV-VP3N88 decreased significantly between 60 and 108 hpi, and was always 10-100-fold lower than that of the parental virus rM9905. The levels of rCAV-VP3N80 were also 10-100-fold lower than that of rM9905 and declined significantly at three time points. There was almost no difference in the viral loads of rCAV-VP3N88 and rCAV-VP3N80. Additionally, rM9905 induced 85.39 ± 2.18% apoptosis at 96 hpi, whereas rCAV-VP3N88 and rCAV-VP3N80 induced 63.08 ± 4.78% and 62.56 ± 7.35% apoptosis, respectively, which were significantly (about 20%) lower than that induced by the parental virus. The rescued viruses altered the nuclear localization in MDCC-MSB1 cells. Moreover, deletion of C-terminal region of apoptin impaired viral replication in vivo and reduced the virulence of CAV in chickens.
  
  
  CONCLUSIONS
  In summary, we have demonstrated that the C-terminal deletion of apoptin in infectious CAV affected the replication of the virus. The deletion of the C-terminal region of apoptin not only significantly reduced viral replication in vitro but also reduced its induction of apoptosis, which correlated with the loss of its nuclear localization. The deletion of the C-terminal region of apoptin also impaired the replication of CAV and attenuated its virulence in chickens.
authors.affiliation: College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, People's Republic of China.|State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Yongqiang
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Xiuqing
authors.initials: X
authors.lastname: Song
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Honglei
authors.initials: H
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, People's Republic of China.
authors.forename: Yonghao
authors.initials: Y
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Yulong
authors.initials: Y
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Xiaole
authors.initials: X
authors.lastname: Qi
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Liting
authors.initials: L
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Huan
authors.initials: H
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Yao
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Han
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, People's Republic of China. hlchen1@yahoo.com.|State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People's Republic of China. hlchen1@yahoo.com.
authors.forename: Hualan
authors.initials: H
authors.lastname: Chen
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D017209:Apoptosis; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D017727:Chicken anemia virus / Q000472:pathogenicity* / Q000502:physiology*; D002645:Chickens; D004252:DNA Mutational Analysis; D005434:Flow Cytometry; D018613:Microscopy, Confocal; D060888:Real-Time Polymerase Chain Reaction; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
chemical_list: D036022:Capsid Proteins; C091717:VP3 protein, Chicken anemia virus; D037521:Virulence Factors
keywords: Apoptin; Apoptosis; Chicken anemia virus; Replication; Virulence