Biology Reference
In-Depth Information
Although the exact mechanism of initiation of HCMV lytic DNA synthesis
remains unknown, much of the emphasis has been focused on the
cis
-acting
sequences within oriLyt and the protein encoded by the UL84 open reading frame
(ORF). UL84 is a unique protein that has no known homolog to any cellular or viral
protein. UL84 is a multifunctional protein that is capable of regulating the tran-
scriptional activation mediated by IE2, and evidence indicates that it is a member
of the DExH/D-box family of proteins. This review will discuss (a) what is known
about the
cis
-acting regions in oriLyt that contribute to DNA synthesis and (b) the
properties of the
trans-
acting factors, specifically UL84, required for lytic DNA
synthesis and how they relate to the regulation and initiation of HCMV lytic DNA
replication.
Essential Region I: IE2-UL84 Responsive Promoter in oriLyt
The HCMV oriLyt region (Fig. 1) was defined according to the smallest fragments
that supported amplification in the transient replication assay. The transient repli-
cation assay involves the transfection of oriLyt-containing plasmids into human
fibroblasts followed by infection with HCMV. Total cellular DNA is harvested and
cleaved with two restriction enzymes. The first cleaves input plasmid and cellular
DNA. The second, DpnI, cleaves methylated (unreplicated) plasmid DNA.
Plasmids propagated in bacterial Dam
+
methylase hosts will add a methyl group at
an adenosine (which is one base within the DpnI recognition sequence). Once
plasmid DNA goes through one round of semiconservative DNA synthesis in
mammalian cells, it then becomes unmethylated, since mammalian cells lack an
adenosine methylase. Replicated DNA is distinguished from input plasmid DNA
based on the sensitivity or resistance to cleavage by DpnI where amplified plasmid
DNA migrates more slowly through an agarose gel due to its larger size. Since
DpnI has a 4-base pair recognition sequence, input unreplicated DNA migrates
toward the bottom of the gel. This powerful assay allowed for the fine mapping of
DNA sequences that are essential or contribute to amplification of oriLyt. These
initial studies defined the oriLyt region between nts 90,504 and 93,930 on the
AD169 genome.
Later studies identified a core domain for oriLyt that contained two essential
regions (I and II). These essential regions were originally defined as those sequences
that could not be mutated or deleted and still retain a functional oriLyt in transient
assays (Zhu et al. 1998). Within essential region I, there are several DNA repeat
elements and a highly prymidine-rich DNA sequence referred to as the Y-block
(Zhu et al. 1998) as well as two 29-base pair repeat sequences that contain several
transcription factor-binding sites. In addition, two IE2 binding sites are also present.
The first is a consensus
cis
repression sequence (CRS) that was shown previously
to interact with IE2 (Cherrington et al. 1991; Arlt et al. 1994; Tsai et al. 1997;
Lashmit et al. 1998; Huang and Chen 2002). This consensus CRS element does not
appear to have a functional role in oriLyt promoter activation/repression or DNA
