CONICET | Buscador de Institutos y Recursos Humanos

Equine Arteritis Virus (EAV) has been shown to induce apoptosis in insect cells and the GP5 protein was associated with this process. In order to further understand and dissect this result, we used the plasmid pCDNA 3.1 to construct a recombinant vector expressing GP5 and used it to transfect mammalian cells. As GP5 forms a heterodimer with M protein, we also constructed a recombinant vector expressing M protein to analyze the possible effect in apoptosis pathway of the latter protein.  The EAV GP5 and M genes were amplified using cDNA from the Bucyrus EAV strain. Two pair of primers with a restriction site sequence were designed to allow the subsequent cloning of these genes into the pCDNA3.1 vector. After checking the correct ligation of both genes to the vector pCDNA3.1, the pCDNA-GP5 and pCDNA-M constructs were transfected separately into Rabbit Kidney (RK13) cells and selected with Geneticin at 400 ug/ml. The pCDNA3.1 with no gene was also transfected as a control of apoptosis by this plasmid backbone.  Activation of caspase 3 was analyzed in situ in RK13 cell line by immunostaining with Amino Ethyl Carbazole (AEC). Staurosporine (1 ug/ml) and RK13 transfected with pCDNA3-1 backbone were used as positive and negative controls, respectively.  EAV-protein detection was done using EAV polyclonal antiserum at 1:50 dilution.  Caspase 3 was not detected in the cells transfected with pCDNA backbone nor in pCDNA-M and pCDNA-GP5. Our results show that although expression of GP5 in insect cells showed evidence of caspase 3 activation expression of GP5 in mammalian cells did not induce such activation. The same result was found for M protein when assayed in mammalian cells. Further investigations need to be done to confirm these results. 