The 2A2 protein of Duck hepatitis A virus type 1 induces apoptosis in primary cell culture.

ABSTRACT

Duck hepatitis A virus type 1, (DHAV-1) 2A2pro, is one of the most highly conserved viral proteins within the DHAV serotypes. However, its effect on host cells is unclear. We predicted that DHAV-1 2A2pro was a GTPase-like protein based on the results of multiple sequence alignment and homologous modeling analysis. Upon transfection of a recombinant plasmid expressing DHAV-1 2A2, cells displayed fragmented nuclei, chromatin condensation, oligonucleosome-sized DNA ladder, and positive terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining; hence, cell death has the characteristics of apoptosis. By staining cells with fluorescein Annexin V-FITC and PI, it is possible to distinguish and quantitatively analyze nonapoptotic cells, early apoptotic cells, late apoptotic/necrotic cells, and dead cells through flow cytometry and fluorescence microscopy. The percentage of apoptotic cells gradually increased and reached a maximum after 48 h of transfection. In conclusion, apoptosis induced by this GTPase-like protein may contribute to DHAV-1 pathogenesis.