Role of Cyclin Dependent Kinases in Viral RNA Processing

Our hypothesis for the altered pattern of expression for the IE1-72/IE2-86 and UL37 IE RNAs in the presence of Roscovitine is based on the phosphorylation of the C-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II) by cdk7/cyclin H and cdk9/cyclin T. Cdk7/cyclin H is responsible for activating cdks1, 2, and 4 and is also a part of TFIIH, which phosphorylates the carboxyl terminal domain (CTD) of the large subunit of RNA polymerase II within the 52 repeats of the heptapeptide YSPTSPS. Cdk9/cyclin T (P-TEFb) also phosphor-ylates the CTD. The current consensus is that transcription and RNA processing are integrated events whereby the differential phosphorylation of the CTD repeats at Ser 2 and Ser 5 defines its affinity for various transcription factors, kinases, and RNA processing factors. When the transcription initiation complex is formed on a 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 contrast, 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 transcription 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 localization 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 concentrate 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 distinct 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

Viral Transcriptosome

Input genome

Cdk9

Fig. 3 Model of the viral transcriptosome formed at the beginning of the infection. The input genome that is deposited at the POD structure functions as the template for IE RNA synthesis. Cellular hypophosphorylated RNAP IIa is recruited to the site along with cdk9 and cdk7, which hyperphosphorylate RNAP IIa to the transcriptionally active RNAP IIo that serves as a platform for RNA processing enzymes. The IE transcripts are synthesized and translated into the IE1-72 and IE2-86 proteins, which return to this nuclear body. IE1-72 causes POD dispersal and it also disperses, while IE2-86 remains at the established transcription site, referred to as the transcripto-some. The inset is an infected cell nucleus at 8 h p.i. and shows the accumulation of both cdk9 and IE2 at several transcriptosomes

—Ser-2 RNAP Ito Poly A Factors

Cdk7

Input genome

Cdk9

Fig. 3 Model of the viral transcriptosome formed at the beginning of the infection. The input genome that is deposited at the POD structure functions as the template for IE RNA synthesis. Cellular hypophosphorylated RNAP IIa is recruited to the site along with cdk9 and cdk7, which hyperphosphorylate RNAP IIa to the transcriptionally active RNAP IIo that serves as a platform for RNA processing enzymes. The IE transcripts are synthesized and translated into the IE1-72 and IE2-86 proteins, which return to this nuclear body. IE1-72 causes POD dispersal and it also disperses, while IE2-86 remains at the established transcription site, referred to as the transcripto-some. The inset is an infected cell nucleus at 8 h p.i. and shows the accumulation of both cdk9 and IE2 at several transcriptosomes beginning of the infection serves as a specialized site for recruitment of cellular and viral proteins necessary for viral transcription and replication. The correct phosphorylation of the RNAP II CTD at these sites is essential for accurate processing of the IE transcripts and for transcription of early genes, and it appears that the required level of phosphorylation is established within the first 8 h.

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