By computer search, we identified one potential NF-kappa B binding site in
the HPV16 long control region (LCR) at position 7554-7563 having two mismat
ches in comparison to the consensus NF-kappa B binding site of the Ig kappa
L promoter. Bandshift experiments with nuclear extracts from HeLa cells or
purified glutathione S-transferase-p65 fusion protein clearly demonstrated
that NF-kappa B is able to bind to this region of the LCR. However, in com
parison to NF-kappa B binding on a consensus probe, the affinity of NF-kapp
a B for this site is about 250-fold reduced. When mutations were introduced
into this NF-kappa B binding site, the activity of the LCR was increased,
strongly suggesting that NF-kappa B was acting as a transcriptional repress
or in the context of the HPV16 LCR. In addition, overexpression of NF-kappa
B p65 repressed the activity of the HPV16 LCR, strengthening this conclusi
on. (C) 2000 Academic Press.