Open AccessMultiple mechanisms are implicated in the regulation of NF-kappa B activity during human cytomegalovirus infection.
Author(s) -
Timothy F. Kowalik,
Bret A. Wing,
J. S. Haskill,
Jane Clifford Azizkhan,
Albert S. Baldwin,
EngShang Huang
Publication year - 1993
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.90.3.1107
Subject(s) - electrophoretic mobility shift assay , enhancer , biology , microbiology and biotechnology , chloramphenicol acetyltransferase , kappa , p50 , gene , nfkb1 , antibody , reporter gene , gene expression , transcription factor , virology , biochemistry , genetics , linguistics , philosophy
Infection-induced activation of the human cytomegalovirus major immediate early enhancer/promoter has been shown to be regulated primarily by transcription factor NF-kappa B cis elements. However, the mechanism(s) by which human cytomegalovirus induces NF-kappa B activity is unknown. A study was therefore undertaken to determine how this virus would affect normal NF-kappa B regulation. Viral infection of fibroblasts resulted in the specific stimulation of promoters containing major histocompatibility complex NF-kappa B cis elements fused upstream of the chloramphenicol acetyltransferase reporter gene. Electrophoretic mobility shift assays of nuclear extracts derived from mock- and virus-infected cells showed dramatic and sustained increases in DNA-binding proteins specific for these NF-kappa B sequences. Experiments using MAD-3 I kappa B, a specific inhibitor of NF-kappa B, and antibodies directed against rel family members demonstrated that the induced binding activities contained p50 and p65 proteins but not c-rel. Northern analysis indicated maximal levels of p50 mRNA by 4 h postinfection, whereas p65 and MAD-3 I kappa B mRNA accumulation peaked at 48-72 h postinfection, suggesting different regulatory mechanisms for p50 and p65/I kappa B genes. Electrophoretic mobility shift assays with deoxycholate-treated cytoplasmic extracts demonstrated a 3- to 4-fold decrease in the cytosolic stores of NF-kappa B binding activity by 4 h postinfection. Western blots probed with antibodies directed against MAD-3 I kappa B or pp40 (a protein isolated from chicken with sequence and biochemical properties similar to those of MAD-3 I kappa B) indicated that a cross-reactive peptide of 39 kDa was no longer detectable after 24 h postinfection. These results demonstrate that the activation and maintenance of nuclear NF-kappa B DNA binding and enhancer activities upon human cytomegalovirus infection occurs by multiple mechanisms.