RSV P-protein impairs extrinsic apoptosis pathway in a macrophage-like cell line persistently infected with respiratory syncytial virus

Abstract

Disabling apoptosis is practically a mandatory step for establishing and maintaining viral persistence in host cells. Thus, persisting viruses have evolved strategies to impair apoptosis mechanisms. Apoptosis can be induced through either the intrinsic or the extrinsic pathway. Previously, we reported that staurosporine-induced intrinsic apoptotic pathway was down-regulated in a macrophage cell line persistently infected with respiratory syncytial virus (RSV, MΦP). In the present study, our results showed that the extrinsic apoptotic pathway was also impaired in this cell line and that RSV P-protein interfered with the onset of the extrinsic apoptotic process. In this work, we analyzed and compared the expression of several components of the DISC complex (i.e., TNF-α, TNFR1, caspase-8, and cIAP2) in MΦP cells with that in mock-infected macrophages. Additionally, by using DNA sequence analysis in silico, we searched for an RSV protein putatively interfering with the triggering of the extrinsic apoptotic process. The analysis showed that viral P-protein shared a 52% homology with the caspase-8 death domain. Subsequently, the nucleic acid sequence of the viral P-protein was cloned and transfected into the macrophage cell line; the effect of this transfection on staurosporine-induced apoptosis was evaluated by assaying for cell viability and caspases-8 and -9 activity. The results revealed that although caspase-9 was activated, the activity of the caspase-8 was impaired in the RSV P-protein transfected cells; more of these cells survived than did mock-transfected cells. These findings suggest that P-protein impaired the extrinsic pathway of apoptosis. Our findings contribute to the understanding of the mechanism by which viral proteins subvert the extrinsic apoptosis process in cells persistently infected with RSV.

Keywords: Extrinsic apoptotic pathway; RSV P-protein; RSV persistence.