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VP2 of Chicken Anaemia Virus Interacts with Apoptin for Down-regulation of Apoptosis through De-phosphorylated Threonine 108 on Apoptin.

ABSTRACT: Chicken anaemia virus (CAV) is an important contagious agent that causes immunosuppressive disease in chickens. CAV Apoptin is a nucleoplasmic shuffling protein that induces apoptosis in chicken lymphoblastoid cells. In the present study, confocal microscopy revealed co-localisation of expressed CAV non-structural protein VP2 with Apoptin in the nucleus of MDCC-MSB1 cells and the nucleoplasmic compartment of CHO-K1 cells. In vitro pull-down and ex vivo biomolecular fluorescent complementation (BiFC) assays further showed that the VP2 protein directly interacts with Apoptin. Transient co-expression of VP2 and Apoptin in MDCC-MSB1 cells significantly decreased the rate of apoptosis compared with that in cells transfected with the Apoptin gene alone. In addition, the phosphorylation status of threonine 108 (Thr108) of Apoptin was found to decrease upon interaction with VP2. Although dephosphorylated Thr108 did not alter the subcellular distribution of Apoptin in the nucleus of MDCC-MSB1 cells, it did suppress apoptosis. These findings provide the first evidence that VP2 directly interacts with Apoptin in the nucleus to down-regulate apoptosis through alterations in the phosphorylation status of the latter. This information will be useful to further elucidate the underlying mechanism of viral replication in the CAV life cycle.

SUBMITTER: Lai GH

PROVIDER: S-EPMC5665943 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundApoptin, a nonstructural protein encoded by the VP3 gene of chicken anemia virus (CAV), has been shown to not only induce apoptosis when introduced into the precursors of chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria.ResultsSignificantly increased expression of recombinant full-length apoptin that originated from chicken anemia virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT) protein transduction domain (PTD). The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptin(opt) in E. coli BL21(DE3) was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25 °C. Moreover, approximately 90% of the expressed GST-TAT-Apoptin(opt) under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptin(opt) protein was used to evaluate the recombinant protein's apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GST-TAT-apoptin(opt) showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to enter apoptosis.ConclusionsOn expression in E. coli, purified recombinant TAT-Apoptin(opt) that has been fused to a GST tag and had its codons optimized, was found to have great potential. This protein may in the future allow the development of a therapeutic protein that is able to specifically kill tumor cells.

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VP2 of Chicken Anaemia Virus Interacts with Apoptin for Down-regulation of Apoptosis through De-phosphorylated Threonine 108 on Apoptin.

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