Many viruses induce cell death and lysis as part of their replication and dissemination strategy, and in many cases features of apoptosis are observed. Attempts have been made to further increase productivity by prolonging cell survival via the over-expression of anti-apoptotic genes. Here, we extend the study to investigate the association between virus replication and apoptosis, pertinent to large-scale vector production for gene therapy. Infection of an HEK293 cell line with a replication defective type-5-adenovirus expressing a GFP reporter (Ad5GFP) resulted in rapid decline in viability associated with increased virus titer. The over-expression of bcl-2 resulted in improved cell resistance to apoptosis and prolonged culture duration, but reduced virus specific and total productivity. In contrast, the over-expression of pro-caspase-3 (Yama/CPP32/apopain) resulted in reduced cell survival but increased virus productivity. The treatment of infected cells with caspase inhibitors support the preposition that caspase-3 dependent apoptosis, and to a lesser degree caspase-9 dependent apoptosis, represent important steps in virus production, thus implicating the intrinsic apoptosis pathway in the production of adenovirus from HEK293 cells. The suppression of apoptosis by the over-expression of XIAP (inhibitors of caspase family cell death proteases) further shows that caspase-mediated activation plays an important role in virus infection and maturation. Biotechnol. Bioeng. 2009;104: 752-765. (C) 2009 Wiley Periodicals, Inc.