Human cytomegalovirus (HCMV), a herpesvirus, is an obligate parasite whose life cycle requires an intricate set of interactions between the virus and the host that optimize the environment for viral replication and assembly (for a review, see reference 14
). Intertwined with this subversion of the host cell is a defined temporal order of viral gene expression that has been loosely divided into three phases—immediate early (IE), early, and late. The IE gene products are synthesized soon after infection and rely primarily on host factors for their expression, although proteins carried in the viral particle clearly contribute to the process. Several of the viral IE proteins serve as essential transactivators of the next class of gene products, the early genes. Included in the early class are those viral proteins required to “activate” the cell to a metabolic state most conducive for viral DNA synthesis and those proteins involved in the actual replication process itself. Late genes, which constitute the majority of the viral genome and encode primarily structural and maturation proteins, are transcribed in abundance only after the onset of viral DNA replication.
Upon infection, the viral DNA enters the nucleus and a subset of HCMV genomes are deposited at nuclear structures referred to variously as nuclear domain 10 (ND10) structures or promyelocytic leukemia protein (PML) oncogenic domains (PODs), where viral RNA synthesis begins (21
). The HCMV tegument protein pp71 interacts with POD-associated Daxx, which may contribute to the initiation of transcription at POD sites (9
). The proximity of the PODs to the spliceosome assembly factor SC35 domains may aid in rapid expression of IE genes, which are often multiply spliced (22
). A major region of viral IE transcription includes two genetic units, IE1 and IE2 (for reviews, see references 15
). The predominant IE RNA (IE1) consists of four exons; a single open reading frame (ORF) (UL123) initiates in exon 2 and specifies a 72-kDa nuclear protein designated IE1-72. The IE2 gene product, IE2-86 (ORF UL122), is an 86-kDa protein that is encoded by an alternatively spliced RNA that contains the first three exons of IE1 and a different terminal exon. Another region of IE gene expression is UL36-38, which includes at least five transcripts directed by three promoters (1
). One of the promoters directs the synthesis of several spliced 3.2- to 3.4-kb RNAs (UL37 and UL37M
ORFs) that are present in small amounts only at IE times as well as an abundant 1.7-kb unspliced RNA that encodes the UL37 exon 1 (UL37X1) gene product.
Newly synthesized IE1-72 and IE2-86 localize to the PODs. While the punctate pattern of the IE2-86 protein persists, at 3 to 6 h postinfection (p.i.), both IE1-72 and POD-associated proteins become dispersed throughout the nucleus (5
). Several studies have shown that IE2-86 is able to localize to the PODs in the absence of IE1-72 but is not able to disrupt them (4
). IE1-72 is required for disruption of the PODs, but since an IE1 deletion mutant virus (CR208) replicates well at high multiplicity (18
), this event is not essential. It appears that even after IE1-72 has caused the dispersal of the PODs, these locations remain important for viral replication. The UL112-113 early gene proteins appear to colocalize with IE2-86 at the peripheries of the original POD sites beginning at about 6 h p.i., and these go on to form sites of viral DNA replication (3
). By 48 h p.i., there is a high level of viral DNA synthesis in the replication centers, and the majority of the viral genome is being transcribed.
Viral transcription is directed by the cellular RNA polymerase II (RNAP II), a multisubunit complex. The largest subunit of RNAP II contains a C-terminal domain (CTD), which in human cells consists of 52 repeats of the consensus heptapeptide sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The CTD is differentially phosphorylated primarily at the serine 2 and serine 5 positions, and the level of phosphorylation varies considerably during the transcription cycle (for reviews, see references 27
, and 37
). Hypophosphorylated RNAP II (RNAP IIa) is recruited to the initiation complex, and the CTD is then phosphorylated to a hyperphosphorylated form (RNAP IIo). The phosphorylation of the serines in position 5 by cdk7/MAT-1/cyclin H (a component of the basal transcription factor complex TFIIH) is followed by the phosphorylation of the serines in position 2 by cdk9/cyclin T (also referred to as positive transcription elongation factor B [P-TEFb]), which is associated with the commitment of the RNAP II complex to elongation. RNAP IIa and IIo forms are in a dynamic equilibrium such that RNAP IIo needs to be dephosphorylated to the IIa form in order to engage in another round of transcription. The primary phosphatase is Fcp1, which is highly conserved and can remove phosphates from the CTD of RNAP IIo when it is engaged in transcription or free. Recently, other phosphatases have been discovered, including the small CTD phosphatases and Ssu72.
The prevailing hypothesis is that the CTD plays a regulatory role in all steps of transcription by serving as the binding domain and transporter of factors involved in RNA synthesis initiation, elongation, 5′ capping, splicing, and cleavage/polyadenylation (for reviews, see references 8
, and 38
). It is believed that the differential recruitment and binding of specific factors that are involved in these processes are significantly influenced by the pattern of phosphorylation of the various Ser2 and Ser5 residues (and possibly by the ubiquitylation, glycosylation, and phosphorylation of other residues) within the 52 repeats. For example, the phosphorylation of the CTD at Ser5 is associated with the recruitment of the mRNA capping enzymes, and the phosphorylation of the CTD at Ser2 is linked to the recruitment of 3′ RNA processing factors.
In a recent study, we used the drug roscovitine, which is a specific inhibitor of the cyclin-dependent kinases (cdk's) 1, 2, 5, 7, and 9 (7
), to examine the role of these kinases in viral replication (44
). We found that addition of the drug at the beginning of the infection resulted in changes in the accumulation and processing of IE transcripts and inhibition of the expression of selected viral early gene products, viral DNA replication, and late gene expression. Roscovitine specifically affected the differential splicing and polyadenylation of the RNAs from both the IE1/IE2 and UL37 genes. The relative position of the sequences used for processing the HCMV unspliced UL37X1 RNA and spliced UL37 IE RNAs (47
) is very similar to the position of the region between UL123 exon 4 and UL122 exon 5, which are used for the alternative cleavage/polyadenylation and splicing that generates the IE1-72 and IE2-86 RNAs, respectively. For both regions, the signals on the RNA for cleavage/polyadenylation overlap those for the downstream 3′ splice acceptor site, with the cleavage/polyadenylation site being preferentially used to generate either IE1-72 or UL37X1 RNAs at IE times. However, in the presence of roscovitine, there was greater utilization of the downstream 3′ splice acceptor site, yielding higher levels of the IE2-86 and spliced UL37 RNAs and corresponding lower levels of the IE1-72 or UL37X1 RNAs. We also showed that when roscovitine was added after the first 4 h of infection, the effects on IE gene expression were no longer observed. When it was added after 6 h, viral replication proceeded through the late phase but the viral titer remained low.
One possible explanation for the altered pattern of viral RNA processing was that the effects of the cdk inhibitor were related to the phosphorylation of the CTD of the large subunit of RNAP II by cdk7/MAT-1/cyclin H and cdk9/cyclin T. A recent paper showing that HCMV induces an intermediate form of phosphorylated RNAP II supports this idea (6
). Those authors proposed that the CTD might be phosphorylated by the HCMV-encoded kinase UL97. However, their data showed that although the CTD can serve as a substrate for UL97 in vitro, RNAP II does not appear to be phosphorylated by this kinase in
In this study, we examine the effect of HCMV infection on the expression, activity, and localization of cdk7/MAT-1/cyclin H, cdk9/cyclin T1, and several forms of the large subunit of RNAP II at both early and late times during the infection. We show that during the course of the infection, there is an increase in cdk7 and cdk9 protein levels and kinase activity and in the amount of RNAP II that is phosphorylated on serine 2 and serine 5 within the CTD. At 48 h p.i., cdk7 and hypophosphorylated RNAP II localize to replication centers, cdk9 and Ser2-phosphorylated RNAP II are distributed in a punctate pattern throughout the nuclei, and Ser5-phosphorylated RNAP II appears in clusters at the peripheries of the viral replication centers. At early times, cdk9 localizes with input viral DNA. In addition, aggregates of cdk9 and cdk7 and a subset of Ser2-phosphorylated RNAP II colocalize with IE1/IE2 proteins adjacent to the PODs. 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. In accord 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. These results suggest that the phosphorylation of the CTD is essential at early time points of the infection and that the required level of CTD phosphorylation for IE gene expression is established within 8 h.