Biology Reference
In-Depth Information
Viral-Encoded
trans
-Acting Factors Required for Lytic
Replication
Most of the information concerning the
trans-
acting factors involved in HCMV
lytic DNA synthesis was elucidated from the cotransfection replication assay. This
assay, which is similar to the assay that elucidated the HCMV cloned oriLyt only
using a set of plasmids that encode putative replication factors, revealed that eleven
loci were required to achieve efficient amplification of cloned oriLyt. The original
assay used plasmid clones that expressed the putative replication proteins under the
control of each of their native promoters. This meant that those factors that contrib-
uted to the efficient expression or activation of replication proteins were also identi-
fied. Therefore the complete set of enzymes involved in the mechanics of DNA
synthesis, as well as any ancillary factors, were identified using this assay (Pari and
Anders 1993; Pari et al. 1993).
Table 1 lists the ORFs identified from the initial cotransfection replication assay
performed in human fibroblasts. Six of these genes identified are common to all
herpesviruses and are designated the core replication proteins, which comprise a
DNA polymerase, polymerase accessory protein, single-stranded DNA binding
protein, helicase, primase and primase associated factor. Interestingly, efficient ori-
Lyt-dependent DNA replication can occur when core proteins from one herpesvi-
rus, for example EBV, is used with a different herpesvirus species oriLyt, for
example HCMV, in the cotransfection replication assay (Sarisky and Hayward
1996; Xu et al. 2004b). In all cases, the only additional factor that is needed is the
initiator protein unique to each herpesvirus. This fact suggests that a set of core
enzymes can carry out DNA synthesis on a primed oriLyt substrate independent of
the mechanism of initiation. It also suggests that the initial events in DNA synthesis
can be performed independent of the enzymes involved in DNA replication, at least
in the context of transient assays.
Table 1
Essential ORFs elucidated from the cotransfection-replication
assay
Open reading frame
Proposed function
UL54
DNA polymerase
UL44
Pol accessory, binds to UL84
UL105
Helicase
UL70
Primase
UL102
Primase-associated factor
UL57
Single-stranded DNA binding
UL84
UTPase, RNA-binding within oriLyt,
regulatory, shuttling protein
IRS1
RNA binding, transactivation
UL112/113
Early proteins, transactivation
UL36-38
Anti-apoptotic
IE2
Transactivation, binds to UL84
