The human cytomegalovirus immediate early 2 protein dissociates cellular DNA synthesis from cyclin-dependent kinase activation

Abstract

Passage through the restriction point late in G1 normally commits cells to replicate their DNA. Here we show that the previously reported cell cycle block mediated by the human cytomegalovirus (HCMV) immediate early 2 (IE2) protein uncouples this association. First, IE2 expression leads to elevated levels of cyclin E-associated kinase activity via transcriptional activation of the cyclin E gene. This contributes to post-restriction point characteristics of IE2-expressing cells. Then these cells fail to undergo substantial DNA replication although they have entered S phase, and the induction of DNA replication observed after overexpression of cyclin E or D can be antagonized by IE2 without impinging on cyclin-associated kinase activities. These data suggest that IE2 secures restriction-point transition of cells before it stops them from replicating their genome. Our results fit well with HCMV physiology and support the view that IE2 is part of a viral activity which, on the one hand, promotes cell cycle-dependent expression of cellular replication factors but, on the other hand, disallows competitive cellular DNA synthesis.

Fig. 1. Cell cycle arrest and deregulation of cdk activities in HCMV-infected U373 cells. Cells were serum starved for 48 h. At the end of the starvation period (0 h) cells were serum restimulated (+ serum), HCMV infected (+ HCMV), or both (+serum, + HCMV). After restimulation/infection, cells were harvested at the indicated time points and aliquots were processed for flow cytometry and cyclin kinase assays. (A) DNA histograms in which the relative DNA content (measured by PI staining) was plotted against cell number. (B) The indicated cyclin complexes were immunoprecipitated (IP) from whole-cell extracts and the associated kinase activities measured with histone H1 (H1) as substrate.

Fig. 2. Physical separation of IE2-expressing cells. U373 cells were transfected with the empty expression vector pSG5-3HA (control) or plasmids expressing p16INK4a (p16), HA-tagged full-length IE2 (IE2) or a HA-tagged IE2 deletion mutant (IE2mut). A CD20 expression vector was included in all transfections in a 1:4 molar ratio. Cells were harvested 48 h after transfection and stained with FITC-anti-CD20 antibody. Following this, cells were separated in fractions enriched for or depleted of CD20-positive cells by anti-CD20-directed MACS. (A) Schematic of IE2 and IE2mut. The amino acids (AA) present in each construct are indicated. AD, transcriptional activation domain; NLS, nuclear localization signal. (B) CD20 expression (measured as FITC fluorescence) of transfected cell populations before or after sorting was plotted against cell size (measured as forward scatter). (C) Equal amounts of extracts from sorted cells were separated by SDS–PAGE and analysed by immunoblotting with an anti-HA antibody. (D) DNA histograms show CD20-positive cell populations in which the relative DNA content (measured by PI staining) was plotted against cell number. The left panel is generated from gated [gate is shown in (B)] but unsorted cells, the right panel from sorted cells [the ‘enriched population’ in (B)]. Data shown in (B), (C) and (D) are from a single experiment representative of multiple cell separations with similar results.

Fig. 3. Analysis of cyclin transcription in IE2-expressing cells. U373 cells were transfected and sorted as described in Figure 2. After isolation of RNAs from sorted cells mRNA expression levels of the indicated cyclins were determined by multiprobe ribonuclease protection assay. As loading controls, probes for L32 and GAPDH were included in each assay.

Fig. 4. Kinase activities of cyclin E, A and B complexes in IE2-expressing cells. U373 cells were transfected and sorted as described in Figure 2. (A) Equal amounts of whole-cell extracts from CD20-positive cells were separated by SDS–PAGE and analysed by immunoblotting with antibodies against cyclins E, A and B. A vigilin-specific antibody was used to control for equal protein loading. A non-specific background band is marked by a star. (B) The same extracts were used for immunoprecipitations (IP) with antibodies against the indicated cyclins followed by kinase assays using histone H1 (H1) as substrate.

Fig. 5. Pocket proteins are hyperphosphorylated in IE2-expressing cells. U373 cells were transfected and sorted as described in Figure 2. Equal amounts of whole-cell extracts from CD20-positive cells were separated by SDS–PAGE and analysed by immunoblotting with antibodies against Rb (A) and p130 (B).

Fig. 6. IE2-mediated inactivation of transcriptional repression by E2F/Rb. U373 cells were transfected and sorted as described in Figure 2. (A) Whole-cell extracts from IE2-arrested (IE2) or cycling (control) cells were incubated with bacterially produced GST–E2F1 bound to glutathione–agarose beads. Input proteins and precipitates from the binding reaction were resolved by SDS–PAGE and analysed by Rb-specific immunoblotting. (B) RNAs of CD20-positive cells were analysed by multiprobe ribonuclease protection assay for mRNA expression levels of E2F and pocket protein family members as indicated. L32 and GAPDH mRNA expression was determined in the same assay for loading control.

Fig. 7. IE2-arrested cells resemble cells at G₁–S transition in their state of cyclin–cdk activity. U373 cells were transfected with the empty expression plasmid pSG5-3HA and pSG5CD20. After removal of the DNA precipitates, cells were treated for 32 h with aphidicolin (Aph) or hydroxyurea (HU) or left untreated (–). After harvesting, cells were sorted as detailed in Figure 2. (A) Cell cycle distributions of CD20-positive, PI-stained cells were analysed by flow cytometry. (B) Kinase activities associated with cyclins E, A and B in extracts of CD20-positive cells were assayed as detailed in the legend to Figure 4B.

Fig. 8. IE2-arrested cells locate at the beginning of S-phase rather than the end of G₁. Cells were transfected with pSG5-3HA or the p16 and IE2 expression plasmids as described in Figure 2. The pSG5-3HA transfected cells were treated with aphidicolin (Aph) or hydroxyurea (HU) as detailed in Figure 7 or left untreated (control). In all transfections BrdU was included in the culture medium after removal of the DNA precipitates. Cells were harvested 32 h later, sorted for CD20 expression and analysed for BrdU incorporation and DNA content by staining with a FITC-labelled BrdU specific antibody and PI, followed by flow cytometry. Shown are density dot plots of CD20-positive cells.

Fig. 9. Cdk-independent arrest of IE2-expressing cells. U373 cells were transiently transfected with pCMVneoBam (control) or vectors expressing cyclin D1 or cyclin E in the absence or presence of an IE2 expression vector. Forty-eight hours after transfection, cells were harvested and sorted for CD20 expression. (A) One aliquot of sorted cells was stained with PI and analysed by flow cytometry. Shown are DNA histograms of CD20-positive cells transfected as indicated. The percentages of cells in G₁ are given. (B) A second aliquot of sorted cells was used for immunoblot analysis with an Rb-specific antibody.