Analysis of the anti-apoptotic activity of four vaccinia virus proteins demonstrates that B13 is the most potent inhibitor in isolation and during viral infection.

Abstract

Vaccinia virus (VACV) is a large dsDNA virus encoding ~200 proteins, several of which inhibit apoptosis. Here, a comparative study of anti-apoptotic proteins N1, F1, B13 and Golgi anti-apoptotic protein (GAAP) in isolation and during viral infection is presented. VACVs strains engineered to lack each gene separately still blocked apoptosis to some degree because of functional redundancy provided by the other anti-apoptotic proteins. To overcome this redundancy, we inserted each gene separately into a VACV strain (vv811) that lacked all these anti-apoptotic proteins and that induced apoptosis efficiently during infection. Each protein was also expressed in cells using lentivirus vectors. In isolation, each VACV protein showed anti-apoptotic activity in response to specific stimuli, as measured by immunoblotting for cleaved poly(ADP ribose) polymerase-1 and caspase-3 activation. Of the proteins tested, B13 was the most potent inhibitor, blocking both intrinsic and extrinsic stimuli, whilst the activity of the other proteins was largely restricted to inhibition of intrinsic stimuli. In addition, B13 and F1 were effective blockers of apoptosis induced by vv811 infection. Finally, whilst differences in induction of apoptosis were barely detectable during infection with VACV strain Western Reserve compared with derivative viruses lacking individual anti-apoptotic genes, several of these proteins reduced activation of caspase-3 during infection by vv811 strains expressing these proteins. These results illustrated that vv811 was a useful tool to determine the role of VACV proteins during infection and that whilst all of these proteins have some anti-apoptotic activity, B13 was the most potent.