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useful as it has high specificity for cdks 1, 2, 5, 7, and 9 and reversibly inhibits
their activity by competing for binding of ATP (De Azevedo et al. 1997). While
the inhibitor has provided several important insights, its broad effects on multiple
cdks necessitates further experiments to determine the roles of individual cdk
complexes during infection. Nevertheless, studies with such chemical inhibitors
are a good starting place for understanding the impact of cellular kinase activity
on virus replication.
Cdk activity appears to be required primarily at two distinct time intervals in the
infection: one during the first 8 h and the second after the onset of viral DNA
replication (Bresnahan et al. 1997; Sanchez et al. 2004; Sanchez and Spector 2006).
Studies from our lab showed a significant decrease in the expression of select early
and late genes and a reduction in viral DNA replication when the drug was added
at the time of infection. Notably, processing of IE transcripts was altered in the
presence of the drug. However, these effects on IE transcript processing and the
inhibition of gene expression and viral DNA replication were not detected if addi-
tion of the drug was delayed until 6-8 h postinfection (p.i.). In contrast, a reduction
of virus titer was observed even if the drug was not added until 48 h p.i.
The effects on IE gene expression when Roscovitine is added at the beginning
of the infection were particularly intriguing and provided the impetus for further
studies. IE1-72 and IE2-86 are alternatively spliced transcripts that share their first
three exons and differ in their 3′ terminal exon, with the 3′ exon of IE2-86 being
most distal from the start site of transcription. At early phases of the infection,
IE1-72 RNA accumulates preferentially compared to IE2-86 RNA; however, as the
infection progresses, more IE2-86 transcripts accumulate, while there is only a
slight increase in IE1-72 transcripts. If Roscovitine is added at the onset of infec-
tion, IE2-86 RNA accumulation is favored at early phases and there is little
increase in the level of IE1-72 transcript (Sanchez et al. 2004). In fact, there is a
decrease in IE1-72 RNA relative to untreated control samples. Given that the
polyadenylation signals for IE1-72 and splicing signals for IE2-86 are juxtaposed,
the most likely explanation for these results is that the differential splicing and
polyadenylation of the UL122-123 transcript is altered by treatment with the drug.
This results in enhanced utilization of the 3′ splice acceptor site that generates the
IE2-86 transcript and a decrease in the cleavage/polyadenylation of the IE1-72
transcript. Consistent with this hypothesis, the differential processing of the IE
UL37 RNAs, which also have signals for polyadenylation and splicing in close
proximity, is similarly affected by cdk inhibition.
The requirement for cdks at later times in the infection is directly related to
formation of infectious viral particles. In a recent paper, we showed that addition
of Roscovitine at 24 h p.i., after the onset of viral DNA synthesis, results in a 1- to
2-log decrease in viral titer (Sanchez and Spector 2006). There is a corresponding
decrease in the amount of viral DNA detected in the supernatant from drug-treated
cells, indicating that there is a defect in the production or release of extracellular
particles. Consistent with this result, we observed changes in the expression, post-
translational modification, and localization of virion structural proteins in
Roscovitine-treated cells. The levels of the IE2-86 and pp150 (UL32) proteins
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