Human Gyrovirus Apoptin shows a similar subcellular distribution pattern and apoptosis induction as the chicken anaemia virus derived VP3/Apoptin

Abstract

The chicken anaemia virus-derived protein Apoptin/VP3 (CAV-Apoptin) has the important ability to induce tumour-selective apoptosis in a variety of human cancer cells. Recently the first human Gyrovirus (HGyV) was isolated from a human skin swab. It shows significant structural and organisational resemblance to CAV and encodes a homologue of CAV-Apoptin/VP3. Using overlapping primers we constructed a synthetic human Gyrovirus Apoptin (HGyV-Apoptin) fused to green fluorescent protein in order to compare its apoptotic function in various human cancer cell lines to CAV-Apoptin. HGyV-Apoptin displayed a similar subcellular expression pattern as observed for CAV-Apoptin, marked by translocation to the nucleus of cancer cells, although it is predominantly located in the cytosol of normal human cells. Furthermore, expression of either HGyV-Apoptin or CAV-Apoptin in several cancer cell lines triggered apoptosis at comparable levels. These findings indicate a potential anti-cancer role for HGyV-Apoptin.

Figure 1

Expression of CAV- and HGyV-Apoptin in cancer cells. (a) Alignment of the protein sequence of CAV-Apoptin (NP_056774.1) and HGyV-Apoptin (CBZ41794.1) using computer software Align (http://xylian.igh.cnrs.fr/bin/align-guess.cgi). Important functional domains, including LRS (leucine-rich domain), NLS1/2 and NES are indicated by boxes and the predicted phosphorylation sites threonine 108 or threonine 111 are indicated by black arrows. Whole cell lysates of HCT116 colon carcinoma (b) and Saos-2 osteosarcoma (c) cells transfected with the indicated plasmids were prepared and western blot analysis for detection of GFP- or FLAG-Apoptin, respectively, as well as β-Actin as a loading control was performed. Molecular weights of the bands are indicated with arrows and blots were cut and combined at the black line

Figure 2

Nuclear translocation of GFP- and FLAG-Apoptin in cancer cells. HCT116 (a) and Saos-2 (b) cancer cells were transfected with pCAV-GFP-AP or pHGyV-GFP-AP and the corresponding pEGFP-C1 control plasmid. Cells were directly imaged in culture dishes after 1 or 2 days (a) or fixed and counterstained with DAPI at 2 and 5 days post-transfection (b) as indicated. Saos-2 cells (c) transfected with pCAV-FLAG-AP or pHGyV-FLAG-AP were fixed after 5 days and stained with a primary mouse anti-FLAG and secondary FITC anti-mouse antibody for detection of FLAG-Apoptin. Nuclei were detected by counterstaining with DAPI. Arrows indicate apoptotic nuclei

Figure 3

Induction of cell death in Saos-2 cells at 5 days after transfection. Saos-2 cells were transfected by nucleofection with pCAV-GFP-AP, pHGyV-GFP-AP or the corresponding pEGFP-C1 control plasmid, as well as pCAV-FLAG-AP and pHGyV-FLAG-AP. Cell death was measured by flow cytometric analysis after Annexin-V-APC/ PI staining. (a) Quantification of cell death as % of APC-labelled cells in the GFP-positive population after 5 days (*P<0.01). Error bars indicate standard deviation of three experiments. (b) Corresponding FACS dot plots of Annexin-V-APC (x-axis) versus PI (y-axis) obtained after 2 and 5 days. (c) After 5 days transfected Saos-2 cells were fixed, stained with a primary mouse anti-FLAG and secondary FITC anti-mouse antibody (for pCAV-FLAG-AP and pHGyV-FLAG-AP) and counterstained with DAPI for the detection of nuclear morphology. Cell death was quantified as the percentage of GFP- or FLAG-positive cells showing condensed or fragmented nuclei. Error bars indicate standard deviation of two independent experiments

Figure 4

Cytosolic expression of GFP-Apoptin and quantification of apoptosis in 1BR3 normal human fibroblasts. Normal (1BR3) and transformed (1BR3LT) human fibroblast cell lines were transfected by nucleofection with pCAV-GFP-AP, pHGyV-GFP-AP or the corresponding pEGFP-C1 control plasmid as well as pCAV-FLAG-AP and pHGyV-FLAG-AP. After 3 days cells were fixed and counterstained with DAPI to analyse the subcellular localisation of GFP-Apoptin. Expression of FLAG-AP was detected using primary mouse anti-FLAG and secondary FITC anti-mouse antibody (a). Apoptosis was quantified by scoring GFP- or FLAG-positive cells containing condensed or fragmented nuclei (b). Over one hundred transfected cells were analysed and experiments were repeated twice (error bars indicate standard deviation)