Biology Reference
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synthesis since it can be deleted without affecting oriLyt amplification. A second
IE2-binding site is nonconventional and was discovered using CASTing and SAAB
methods (Huang and Chen 2002). This second site appears to be functional in that
it was shown to interact with HCMV IE2 (IE86) in vivo using the chromatin immu-
noprecipitation assay (ChIP) (Xu et al. 2004b). In addition, the DNA sequence
containing the nonconventional IE2-binding site was shown to have promoter
activity, driving the expression of a luciferase reporter upon HCMV infection, or in
the presence of UL84 and IE2 in transient assays in HFF cells (Xu et al. 2004b).
The nonconventional IE2-binding site does contain a CRS-like element that is simi-
lar to those found in other IE2-responsive promoters, and this DNA motif was
shown to be required for efficient promoter activity and oriLyt amplification. In the
context of oriLyt, it appears that this promoter element may participate in the initial
events surrounding the regulation of DNA synthesis and could serve as the trigger
for initiation of lytic DNA synthesis. For example, one such scenario could be that
when UL84 protein is produced it interacts with IE2 and facilitates the binding of
the protein complex to oriLyt, thereby initiating transcription and signaling the
onset of lytic DNA replication.
The presence of an IE2-UL84 responsive promoter suggested that transcription
plays an important role in the activation of lytic DNA synthesis. This is not unlike
other herpesviruses such as EBV and HHV-8, where promoter elements are part of
lytic origins and their presence is essential for efficient oriLyt activity
(Hammerschmidt and Sugden 1988; Aiyar et al. 1998; Lin et al. 2003; AuCoin
et al. 2004; Wang et al. 2004). The HCMV oriLyt promoter element extends from
AD169 nts 91495-92888, and appears to be bidirectional in that transcription
driven in either direction can accommodate oriLyt amplification. Transient reporter
assays demonstrated that either orientation of the oriLyt promoter region could
drive the expression of luciferase. The activity of this promoter is cell type-
dependent since transient transfection of luciferase reporter plasmids containing
the oriLyt promoter were constitutively active in Vero cells, whereas transcrip-
tional activation in human fibroblasts (HFs) was only achieved by subsequent viral
infection or the cotransfection of IE2 and UL84. The constitutively active oriLyt
promoter in Vero cells led to the observation that IE2 was not required for oriLyt
amplification in the cotransfection replication assay in those cells (Sarisky and
Hayward 1996). In HFs however, amplification of oriLyt requires both IE2 and
UL84, presumably to activate the oriLyt promoter. Interestingly, when the cotrans-
fection replication assay was performed in HFs constitutively expressing the cata-
lytic subunit of telomerase, these life-expanded cells supported oriLyt amplification
without the need for the proteins expressed from the UL36-38 loci. This suggests
that in the context of the cotransfection replication assay, ORFs UL36-38 may
serve to enhance cell survival.
In an effort to further define the role of transcription in HCMV oriLyt activation,
a strong constitutive promoter was inserted in oriLyt in place of the defined oriLyt
promoter (Xu et al. 2004b) in a manner similar to that done with the EBV oriLyt
(Hammerschmidt and Sugden 1988). The SV40 early promoter was able to func-
tionally substitute for the HCMV oriLyt promoter in transient assays and alleviated
