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J Virol. 2018 Apr 27;92(10). pii: e00238-18. doi: 10.1128/JVI.00238-18. Print 2018 May 15.

Caspase-Dependent Apoptosis Induction via Viral Protein ORF4 of Porcine Circovirus 2 Binding to Mitochondrial Adenine Nucleotide Translocase 3.

Author information

1
Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, China.
2
MOE International Joint Collaborative Laboratory for Animal Health and Food Safety, Institute of Immunology, Nanjing Agricultural University, Nanjing, China.
3
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
4
Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China.
5
College of Veterinary Medicine, China Agricultural University, Beijing, China.
6
Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, China jyzhou@zju.edu.cn.
7
Collaborative Innovation Center, First Affiliated Hospital, Zhejiang University, Hangzhou, China.
8
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou, China.

Abstract

Apoptosis is an essential strategy of host defense responses and is used by viruses to maintain their life cycles. However, the apoptotic signals involved in virus replication are poorly known. In the present study, we report the molecular mechanism of apoptotic induction by the viral protein ORF4, a newly identified viral protein of porcine circovirus type 2 (PCV2). Apoptosis detection revealed not only that the activity of caspase-3 and -9 is increased in PCV2-infected and ORF4-transfected cells but also that cytochrome c release from the mitochondria to the cytosol is upregulated. Subsequently, ORF4 protein colocalization with adenine nucleotide translocase 3 (ANT3) was observed using structured illumination microscopy. Moreover, coimmunoprecipitation and pulldown analyses confirmed that the ORF4 protein interacts directly with mitochondrial ANT3 (mtANT3). Binding domain analysis further confirmed that N-terminal residues 1 to 30 of the ORF4 protein, comprising a mitochondrial targeting signal, are essential for the interaction with ANT3. Knockdown of ANT3 markedly inhibited the apoptotic induction of both ORF4 protein and PCV2, indicating that ANT3 plays an important role in ORF4 protein-induced apoptosis during PCV2 infection. Taken together, these data indicate that the ORF4 protein is a mitochondrial targeting protein that induces apoptosis by interacting with ANT3 through the mitochondrial pathway.IMPORTANCE The porcine circovirus type 2 (PCV2) protein ORF4 is a newly identified viral protein; however, little is known about its functions. Apoptosis is an essential strategy of the host defense response and is used by viruses to maintain their life cycles. In the present study, we report the molecular mechanism of the apoptosis induced by the ORF4 protein. The ORF4 protein contains a mitochondrial targeting signal and is an unstable protein that is degraded by the proteasome-dependent pathway. Viral protein ORF4 triggers caspase-3- and -9-dependent cellular apoptosis in mitochondria by directly binding to ANT3. We conclude that the ORF4 protein is a mitochondrial targeting protein and reveal a mechanism whereby circovirus recruits ANT3 to induce apoptosis.

KEYWORDS:

ANT3; ORF4; mitochondrial apoptosis pathway; porcine circovirus; viral protein

PMID:
29491154
PMCID:
PMC5923066
DOI:
10.1128/JVI.00238-18
[Indexed for MEDLINE]
Free PMC Article

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