Biology Reference
In-Depth Information
the need for IE2, but not UL84, in the replication assay. This strongly suggested
that transcription somehow triggers initiation of DNA synthesis. In addition, these
experiments identified UL84 as the factor most likely to act as an initiator protein
and, since the leftward region of oriLyt is a promoter, the rightward region must
contain a substrate for direct or indirect UL84 interaction.
Essential Region II: RNA/DNA Hybrid Structure
In addition to the IE2-UL84 responsive promoter region found in essential region I,
the downstream region of oriLyt contains RNA/DNA hybrid structures. These
structures were discovered while investigating the presence of alkali-labile regions
within the HCMV genome. This RNA/DNA hybrid region stretches from approxi-
mately the Not I site (nts 92888) to 300 bp upstream of the BamHI site (93513)
(Prichard et al. 1998). Interestingly, this region of the origin is variably reiterated
via tandem duplication in the laboratory strain AD169, resulting in about 300 bp
of extra sequence in the viral genome. The RNA/DNA hybrids are present within
the packaged viral genome and appear to be a stable part of the genome. The RNA/
DNA hybrid region contains several G+C-rich repeat sequences, one of which has
the capacity to form a stem loop configuration. Since this region can be repeated
many times within the viral genome, this stem loop may also be amplified.
Although the significance of the RNA/DNA hybrid and the stem loop remains to
be seen, it has been postulated that essential region II may be the area of oriLyt
that interacts directly with UL84. In vitro RNA binding assays determined that
UL84 does interact with a synthetic RNA stem loop oligonucleotide that is the
same sequence as that present within the RNA/DNA hybrid region of oriLyt. In
addition, UL84 appears to change the conformation of this oligonucleotide in that
the UL84-RNA stem loop complex migrates faster in a native gel when more
UL84 protein is added. This yields a staircase pattern of binding observed only
when RNA stem loop oligonucleotide substrates are used in the binding assay
(Colletti et al. 2007).
With respect to UL84 interactions with oriLyt in the infected cell environment,
our laboratory used the ChIP assay to demonstrate that UL84 does interact with the
RNA/DNA hybrid region of oriLyt in infected cells, as well as in packaged virions.
In addition, we demonstrated that UL84 interacts directly or indirectly with regions
of oriLyt that contain a CAAT enhancer-binding site (C/EBP
α
). This interaction
suggests that UL84 may cooperate with C/EBP
or use these consensus sequences
to facilitate DNA synthesis. This scenario would be consistent with HHV-8 where
K-bZIP interacts with C/EBP
α
and binds to these transcription factor-binding sites
within HHV-8 oriLyt (Wang et al. 2003). Another plausible scenario is that UL84
uses the consensus C/EBP
α
transcription
factor interacting with oriLyt. Although ChIP results do show C/EBP
α
binding sites independent of the C/EBP
α
α
binding to
this region, we were unable to detect a UL84-C/EBP
α
protein interaction within
infected cells.
