Simian virus 5 (SV5) is a member of the paramyxovirus family, which includes emerging viruses such as Hendra virus and Nipah virus as well as many important human and animal pathogens that have been known for years. SV5 encodes eight known viral proteins, including a small hydrophobic integral membrane protein (SH) of 44 amino acids. SV5 without the SH gene (rSV5deltaSH) is viable, and growth of rSV5deltaSH in tissue culture cells and viral protein and mRNA production in rSV5deltaSH-infected cells are indistinguishable from those of the wild-type SV5 virus. However, rSV5deltaSH causes increased cytopathic effect (CPE) and apoptosis in MDBK cells and is attenuated in vivo, suggesting the SH protein plays an important role in SV5 pathogenesis. How rSV5deltaSH induces apoptosis in infected cells has been examined in this report. Tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, was detected in culture media of rSV5deltaSH-infected cells. Apoptosis induced by rSV5deltaSH was inhibited by neutralizing antibodies against TNF-alpha and TNF-alpha receptor 1 (TNF-R1), suggesting that TNF-alpha played an essential role in rSV5deltaSH-induced apoptosis in a TNF-R1-dependent manner. Examination of important proteins in the TNF-alpha signaling pathway showed that p65, a major NF-kappaB subunit whose activation can lead to transcription of TNF-alpha, was first translocated to the nucleus and was capable of binding to DNA and then was targeted for degradation in rSV5deltaSH-infected cells while expression levels of TNF-R1 remained relatively constant. Thus, rSV5deltaSH induced cell death by activating TNF-alpha expression, possibly through activation of the NF-kappaB subunit p65 and then targeting p65 for degradation, leading to apoptosis.