H. Arlt et al., IDENTIFICATION OF BINDING-SITES FOR THE 86-KILODALTON IE2 PROTEIN OF HUMAN CYTOMEGALOVIRUS WITHIN AN IE2-RESPONSIVE VIRAL EARLY PROMOTER, Journal of virology, 68(7), 1994, pp. 4117-4125
The 86-kDa IE2 protein (IE86) of human cytomegalovirus (HCMV) can act
as both an activator and a repressor of gene expression. The mechanism
s for both of these functions are not well defined. It has recently be
en demonstrated that this protein has sequence specific DNA binding pr
operties: it interacts directly with a target sequence that is located
between the TATA box and the cap site of its own promoter. This seque
nce, termed the CRS (cis repression signal) element, is required for n
egative autoregulation of the IE1/IE2 enhancer/promoter by IE2. We dem
onstrate now that binding of this protein to DNA is not confined to th
is site but occurs also within an early promoter of HCMV that has prev
iously been shown to be strongly IE2 responsive. By DNase I protection
analysis using a purified, procaryotically expressed IE2 protein, we
could identify three binding sites within the region of -290 to -120 o
f the UL112 promoter of HCMV. Competition in DNase I protection experi
ments as well as gel retardation experiments showed that the identifie
d binding sites are specific and have high affinity. Deletion of IE2 b
inding sites from this promoter reduced the level of transactivation;
however, the remaining promoter could still be stimulated about 40-fol
d. Constructs in which IE2 binding sites were fused directly to the TA
TA box of the UL112 promoter did not reveal a significant contribution
of these sequences to transactivation. However, if an IE2 binding sit
e was reinserted upstream of nucleotide -117 of the UL112 promoter, an
increase in transactivation by IE2 was obvious, whereas a mutated seq
uence could not mediate this effect. This finding suggests that DNA-bo
und IE2 can contribute to transactivation but seems to require the pre
sence of additional transcription factors. Moreover, a comparison of t
he detected IE2 binding sites could not detect a strong homology, sugg
esting that this protein may be able to interact with a broad spectrum
of different target sequences.