Caspase-dependent apoptosis induction via viral protein ORF4 of porcine circovirus 2 binding to mitochondrial adenine nucleotide translocase 3
Lin C, Gu J, Wang H, Zhou J, Li J, Wang S, Jin Y, Liu C, Liu J, Yang H, Jiang P, Zhou J
J Virol. 2018 Feb 28. pii: JVI.00238-18. doi: 10.1128/JVI.00238-18.
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 viral protein ORF4, a newly identified viral protein of porcine circovirus type 2 (PCV2). Apoptosis detection revealed that not only is the activity of caspases 3 and 9 is increased in PCV2-infected and ORF4-transfected cells, but also cytochrome c release from the mitochondria to the cytosol is upregulated. Subsequently, ORF4 colocalization with adenine nucleotide translocase 3 (ANT3) was observed using structured illumination microscopy. Moreover, co-immunoprecipitation and pulldown analyses confirmed that ORF4 interacts directly with mitochondrial ANT3. Binding domain analysis further confirmed that N-terminal residues 1 to 30 of ORF4, comprising a mitochondrial targeting signal, are essential for the interaction with ANT3. Knockdown of ANT3 markedly inhibited the apoptotic induction of both ORF4 and PCV2, indicating that ANT3 plays an important role in ORF4-induced apoptosis during PCV2 infection. Taken together, these data indicated that ORF4 is a mitochondrial targeting protein that induces apoptosis by interacting with ANT3 through the mitochondrial pathway.IMPORTANCE Porcine circovirus type 2 (PCV2) encoded 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 ORF4. ORF4 contains a mitochondrial targeting signal and is an unstable protein that is degraded by the proteasome-dependent pathway. Viral protein ORF4 triggers caspases-3 and -9-dependent cellular apoptosis in mitochondria by directly binding to ANT3. We concluded that ORF4 is a mitochondrial targeting protein and revealed a mechanism whereby circovirus recruits ANT3 to induce apoptosis.