Biology Reference
In-Depth Information
Introduction
Human cytomegalovirus lytic DNA replication appears to be a complex and highly
regulated event. Although immediate early gene expression occurs shortly after
infection, the onset of viral DNA synthesis does not take place until approximately
24 h postinfection in cell culture. HCMV DNA replication is thought to involve
circularization and concatemer formation (McVoy and Adler 1994). Viral DNA
synthesis, which occurs in the nucleus, requires the HCMV core replication
machinery and may be regulated by multifunctional immediate early proteins that
were originally thought to have a role only in gene regulation. As for the structure
of replicating viral DNA, head-to-tail concatemers are evident from examination of
the replication products from cloned oriLyt in human fibroblasts. Although a roll-
ing circle model is postulated for lytic DNA replication, there is limited evidence
to support this method for HCMV DNA synthesis and actual events may be more
complex (McVoy and Adler 1994).
The
cis
-acting lytic origin of replication, oriLyt, is located within the unique
long (U
L
) region of the genome between AD169 nucleotides 90,500 and 93,930.
This region, which is situated between ORFs 57 and 69, was originally determined
to contain an origin based on the results of both the transient replication assay,
where cloned regions of the HCMV genome were amplified upon transfection and
subsequent infection of permissive cells (Anders and Punturieri 1991; Anders
et al. 1992; Masse et al. 1992), and a method that took advantage of the chain
termination effects of the drug ganciclovir (Hamzeh et al. 1990). Later studies
confirmed that the oriLyt identified form both studies was the only functional
replicator in the virus genome (Borst and Messerle 2005). Both approaches
confirmed that a complex DNA region within the HCMV genome was responsi-
ble for the propagation of viral DNA during the lytic phase of viral growth. With
the cloned lytic origin in hand, a cotransfection replication assay was developed
in an effort to elucidate the viral-encoded
trans
-acting factors that contribute to
oriLyt-dependent DNA replication. The transient cotransfection replication
assay revealed that eleven loci are required for efficient oriLyt-dependent DNA
replication. These genes are: UL36-38, UL44 (pol accessory protein), UL54
(DNA polymerase), UL57 (single-stranded DNA binding protein), UL70 (pri-
mase), UL102 (primase-associated factor), UL105 (helicase), UL112/113 (early
proteins), IRS1/TRS1 (immediate early RNA-binding), IE1/2 (transactivator)
and UL84. Several of these proteins were determined to play ancillary roles in
DNA replication (Sarisky and Hayward 1996; Xu et al. 2004b).
Initiation of lytic DNA replication occurs at a single origin of replication and
appears to be initially mediated by the viral encoded proteins UL84 and IE2.
As our understanding of HCMV lytic DNA matures, it is clear that viral encoded
proteins may function in a regulatory as well as enzymatic role such as recognition
and unwinding of a distinct structure within oriLyt. What this suggests is that rep-
lication proteins themselves may serve to control the onset of DNA synthesis by
interacting with, or acting as
trans-
acting factors themselves.
