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promoter, the CTD of RNAP II is unphosphorylated (the polymerase is designated
RNAP IIa). Cdk7 with cyclin H and MAT1 primarily phosphorylates Ser 5 on the
CTD (the polymerase is now designated RNAP IIo), which leads to the recruitment
of the RNA capping enzymes. Further phosphorylation of the CTD Ser 2 residues
by cdk9/cyclin T occurs upon entry to elongation and is associated with recruitment
of the cleavage/polyadenylation and splicing machinery.
Previously, our lab showed that infected cells contain more cdk7, MAT-1,
cdk9, and cyclin T1 at 24 h p.i. than the uninfected cells, and the abundance and
activity of these proteins increase as the infection proceeds (Tamrakar et al.
2005). All of MAT-1 is complexed with cdk7, although free cdk7 is also present,
and most of cdk9 and cyclin T1 are in complex. In accord with the increase in
the activity of the cdk9 and cdk7 kinases, an increase in the phosphorylation of
the RNAP II CTD, particularly on the Ser 2 and Ser 5 residues of the heptad
repeats, was also noted. By immunofluorescence analysis, it was observed that
cdk7 and hypophosphorylated RNAP II localize to replication centers. In con-
trast, cdk9 and ser2-phosphorylated RNAP II are distributed in a punctate pattern
throughout the nucleus with some concentration at the periphery of the viral
replication centers. Similarly, ser5-phosphorylated RNAP II appears in clusters
at the rim of the viral replication centers. These results suggest that at late times
in the infection, HCMV may commandeer the RNA polymerase machinery, with
viral RNA synthesis initiated within the replication center and active viral tran-
scription occurring at the periphery.
Our lab has also demonstrated that addition of the cdk inhibitor Roscovitine at
the time of infection results in decreased CTD phosphorylation in the infected cells
and a decrease in the level of the hypophosphorylated RNAP II in both infected and
mock-infected cells (Tamrakar et al. 2005). Consistent with our previous results
regarding the effect of the cdk inhibitors on the processing and accumulation of the
HCMV IE1/IE2 and UL37 IE transcripts, the decrease in CTD phosphorylation
does not occur if the drug is added after 8 h p.i. One clue to explain this restricted
interval in which cdk activity is required may be found in the differential localiza-
tion of cdk9 and cdk7 at the beginning of the infection. Upon cell entry, incoming
HCMV genomes localize near ND10, where viral IE transcription begins (Ishov
and Maul 1996; Ishov et al. 1997) (see the chapter by G. Maul, this volume).
Following translation, the IE1-72 and IE2-86 proteins return to nucleus and concen-
trate near the ND10. IE1-72 mediates the dispersal of ND10 associated proteins and
it also disperses, while IE2-86 persists at the site (Kelly et al. 1995; Korioth et al.
1996; Ahn and Hayward 1997; Ishov et al. 1997; Ahn et al. 1998). We find that as
early as 4 h p.i., several proteins involved in RNA transcription, including cdk9,
cdk7, and Ser2-phosphorylated RNA polymerase II, colocalize with IE2-86 in dis-
tinct aggregates (referred to as viral transcriptosomes) adjacent to the ND10 that are
undergoing dispersal (Tamrakar et al. 2005) (see Fig. 3). However, if Roscovitine
is added at the beginning of the infection, IE2-86 is found in the transcriptosome,
but cdk7 and cdk9 are not recruited (Kapasi and Spector 2008). In contrast, both
cdks colocalize with IE2-86 in the transcriptosome if Roscovitine is added after 8 h
p.i. These results suggest that the formation of a distinct viral transcriptosome at the
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