Wild-type herpes simplex virus type 1 (HSV-1) infection triggers apoptosis in human cells. The subsequent synthesis of infected cell proteins between 3 and 6 h postinfection (hpi) acts to block this process from killing the cells. The factors produced during this window also prevent cell death induced by environmental staurosporine or sorbitol (M. Aubert, J. O'Toole, and J. A. Blaho, J. Virol. 73:10359-10370, 1999). We now report that (i) during the prevention window, HSV-1(F) also inhibited apoptosis induced by tumor necrosis factor alpha (TNF-alpha) plus cycloheximide (CHX) treatment. While deciphering the mechanism of this inhibition, we observed that (ii) the transcription factor NF-kappaB translocated from the cytoplasm into the nuclei of infected cells, and (iii) this migration initiated at 3 hpi. (iv) The complete inhibition of protein synthesis at 3 hpi by the addition of CHX precluded NF-kappaB translocation, while CHX additions at 6 hpi or later did not elicit this effect. This result confirms that infected cell protein synthesis is required for the nuclear import of NF-kappaB. (v) The detection of NF-kappaB in nuclei correlated with the ability of HSV-1(F), HSV-1(KOS1.1), or HSV-1(R7032), a replication-competent recombinant virus containing a deletion in the gene encoding the gE glycoprotein, to prevent apoptosis. (vi) NF-kappaB did not bind its kappaB DNA recognition site and remained cytoplasmic in cells actively undergoing apoptosis following infection with HSV-1(vBSdelta27), a virus with the key regulatory protein ICP27 deleted. (vii) Prestimulation of NF-kappaB by the addition of a phorbol ester prevented HSV-1(vBSdelta27)-induced apoptosis. (viii) Retention of NF-kappaB in the cytoplasm by the addition of a pharmacological antagonist of its release from IkappaBalpha led to an increase in death factor processing during HSV-1(F) infection. (ix) A novel HEp-2 clonal cell line, termed IkappaBalphaDN, was generated which expresses a dominant-negative form of IkappaBalpha. Treatment of IkappaBalphaDN cells with TNF-alpha in the absence of CHX resulted in apoptotic death due to the inability of NF-kappaB to become activated in these cells. Finally, (x) infection of IkappaBalphaDN cells with HSV-1(F) or HSV-1(KOS1.1) resulted in apoptosis, demonstrating that (xi) the nuclear translocation of NF-kappaB between 3 and 6 hpi (the prevention window) is necessary to prevent apoptosis in wild-type HSV-1-infected human HEp-2 cells.