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Abstract: Expression of the major immediate-early (MIE) genes of human cytomegalovirus (HCMV) in the human thyroid papillary carcinoma cell line TPC-1 is repressed at the transcriptional level. However, treatment of these cells with hexamethylene bisacetamide (HMBA), a chemical inducer of differentiation, for 12 to 24 h before infection enabled the cells to support IE1 and IE2 gene expression and consequently HCMV replication. In HMBA-treated cells the transcription factor NF-κB was induced and the MIE promoter (MIEP) was activated. The presence of a NF-κB inhibitory peptide SN-50 or expression of a dominant negative IκBα protein during the HMBA pretreatment period efficiently prevented the HMBA-induced MIEP activation and MIE protein synthesis. Moreover, introduction of mutations into the NF-κB binding sites in the MIEP in a plasmid expressing the IE1 protein diminished its ability to express the protein in HMBA-treated cells. Therefore, the NF-κB activity previously induced in HMBA-treated cells and the NF-κB sites in the MIEP were shown to be essential for HCMV to respond to HMBA action and to express the MIE genes. Investigation of the mechanisms by which HMBA activates NF-κB revealed that degradation of IκBα and translocation of the phosphorylated NF-κB p65 subunit to the nucleus, both of which are known to be critical steps in NF-κB activation, are stimulated in the HMBA-treated cells. These results indicate that treatment of nonpermissive TPC-1 cells with HMBA induces MIE gene permissiveness by up-regulating NF-κB activity. [Copyright &y& Elsevier]