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.

Keywords: Apoptin; Apoptosis; Chicken anemia virus; Replication; Virulence.

Fig. 1

Schematic representation of CAV VP3 with different deletions and changes to VP2 (not drawn to scale). a A leucine-rich sequence (LRS) is located at residues 33–46. Nuclear localization signal 1 (NLS1) is located at residues 82–88. Nuclear export signal (NES) is located at residues 97–105. NLS2 is located at residues 111–121. Rescued virus rCAV-VP3N88 is deleted at NES–NLS2 and rescued virus rCAV-VP3N80 is deleted at NLS1–NES–NLS2. b The stop codon of ‘UGA’ is introduced in VP3 to construct rCAV-VP3N88. However, this stop codon will not introduced into VP2 because they are in different frames. And only making an amino acid substitution of ‘A’ with a ‘V’ in VP2. Similarly, the stop codon of ‘UAA’ is introduced in VP3 to construct rCAV-VP3N80. And resulting an amino acid substitution of ‘S’ with a ‘L’ in VP2. These VP3 truncated mutant viurses express the full length VP2 as the parental virus

Fig. 2

Immunofluorescence assay of the rescued viruses. MDCC-MSB1 cells were infected with the parental virus rM9905 (a) or rescued viruses rCAV-VP3N88 (b) and rCAV-VP3N80 (c), or were mock-infected (d). Cells were treated with primary antibody directed against VP3 and FITC-conjugated secondary antibody, and detected with microscopy (×200)

Fig. 3

Viral proteins VP2 and VP3 expressed in MDCC-MSB1 cells infected with rCAV-VP3N88, rCAV-VP3N80, or parental virus rM9905, respectively. Whole cell lysates were separated by SDS-PAGE and transferred to pure nitrocellulose blotting membranes. Monoclonal antibodies against VP2 or VP3 were used as primary antibody, followed by HRP-conjugated Anti-Mouse secondary antibody. Proteins were visualized by ECL assay

Fig. 4

Replication of rescued viruses rCAV-VP3N88 and rCAV-VP3N80 compared with parental strain rM9905 in MDCC-MSB1 cells. MDCC-MSB1 cells were inoculated with the same amounts of virus, quantified with quantitative PCR. Viral replication was determined with quantitative PCR at 48, 60, 72, 84, 96, and 108 h postinfection. The error bars represent the standard deviations at each time point for triplicate assays. Significance of the variations between groups was determined with a t test (#p > 0.05; *p < 0.05; **p < 0.01)

Fig. 5

Localization of rescued viruses rCAV-VP3N88 and rCAV-VP3N80 compared with parental strain rM9905 in MDCC-MSB1 cells. MDCC-MSB1 cells were infected with rescued virus rCAV-VP3N88 or rCAV-VP3N80 or parental virus rM9905. Mock-infected cells were used as the negative control. Viruses were stained with FITC-conjugated antibody (green) and nuclei with propidium iodide (PI; red). The distribution of apoptin was observed with confocal microscopy. Scale bar is shown at the bottom right (10 μm)

Fig. 6

Apoptosis induced by rescued viruses rCAV-VP3N88 and rCAV-VP3N80 compared with that induced by parental strain rM9905 in MDCC-MSB1 cells. MDCC-MSB1 cells were infected with rescued virus rCAV-VP3N88 or rCAV-VP3N80 or parental virus rM9905. At 48, 72, and 96 h postinfection, apoptosis was detected with an annexin V–FITC apoptosis detection kit and flow cytometry. Error bars represent the standard deviations at each time point for triplicate assays. Significance of the variations between groups was determined with a t test (#p > 0.05; *p < 0.05; **p < 0.01)

Fig. 7

Replication of rescued viruses rCAV-VP3N88 and rCAV-VP3N80 compared with parental strain rM9905 in thymuses of SPF chickens. SPF chickens were inoculated with rescued virus rCAV-VP3N88 or rCAV-VP3N80 or parental virus rM9905. The viral replication curve was constructed from the viral DNA loads in the thymus, measured with quantitative PCR. Error bars represent the standard deviations at each time point for triplicate assays. Significance of the variations between groups was determined with a t test (#p > 0.05; *p < 0.05; **p < 0.01)

Fig. 8

Gross changes in the thymuses and spleens of SPF chickens infected with rM9905 (a), rCAV-VP3N88 (b), rCAV-VP3N80 (c), or mock-infected (d)

Fig. 9

Histological changes in the thymuses of SPF chickens infected with rM9905 (a), rCAV-VP3N88 (b), rCAV-VP3N80 (c), or mock-infected (d)