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Instituto Gulbenkian de Ciência, Apartado 14, Oeiras, Portugal
Correspondence
R. M. E. Parkhouse
parkhous{at}igc.gulbenkian.pt
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Izumiya et al., 2003; Jean et al., 2001; Lu & Shenk, 1999). A number of viruses target the critical regulators of G2/M progression, the cyclins and cyclin-dependent kinases or their regulators (Friborg et al., 1999; Groschel & Bushman, 2005; He et al., 1995; Park et al., 2000). The expression of cyclin B oscillates throughout the cell cycle, beginning to accumulate during S phase and reaching its maximum at the G2/M phase. Activation of the Cdc2–cyclin B complex for entry into mitosis is regulated by dephosphorylating Cdc2 on Thr-14 and Tyr-15. In this work, we describe a ‘non-assigned’ gene of murine gammaherpesvirus 68 (MHV68), which has evolved into a host-modifying gene causing inactivation of the Cdc2–cyclin B complex.
MHV68 is a particularly useful laboratory model for the study of gammaherpesviruses such as Epstein–Barr virus and Kaposi’s sarcoma associated herpesviruses (Nash et al., 2001; Simas & Efstathiou, 1998). Originally isolated from wild rodents, it is capable of infecting laboratory strains of mice, thus providing a system to study gammaherpesvirus infection and pathogenesis (Sunil-Chandra et al., 1992a). After intranasal infection, MHV68 establishes a productive infection in lung epithelial cells followed by a latent infection of spleen B cells, macrophages, dendritic cells and lung epithelial cells (Flano et al., 2000; Sunil-Chandra et al., 1992b; Weck et al., 1999). In addition, MHV68 replicates efficiently in vitro, thereby permitting the study of early events in virus infection and replication.
Although the genome of MHV68 contains genes unique to this virus (Virgin et al., 1997), there are many others that are conserved among the herpesvirus subfamilies. The majority of these have identified functions, but there are a significant number of shared, non-homologous herpesvirus genes without a known function. One of these is ORF20 from MHV68, which has a predicted N-terminal domain homologous to herpesvirus UL24 gene products (Alba et al., 2001). The universal presence of UL24 in herpesviruses genomes from the three subfamilies, alpha-, beta- and gammaherpesviruses, suggests a fundamental and conserved role in the biology of herpesviruses.
Here, we have demonstrated that, as reported for its human herpesvirus homologues, ORF20 localizes in the nucleus. More importantly, expression of MHV68 ORF20 induced G2/M cell-cycle arrest followed by apoptosis of both mouse and human cells. The mechanism of its action is to maintain Cdc2 in its phosphorylated inactive state, so that Cdc2–cyclin B complexes in cells expressing ORF20 exhibit almost no kinase activity.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Plasmids.
MHV68 ORF20 was cloned into plasmid pCDNA3 fused in frame with an N-terminal influenza haemagglutinin (HA) peptide tag. The human cytomegalovirus (CMV)–vesicular stomatitis virus G envelope protein, the packaging plasmid pCMVR8.9 and the vector pHR-CMV-eGFP constructs have been described previously (Ikeda et al., 2002).
For construction of a recombinant lentivirus vector (pHR-CMV-ORF20eGFP), ORF20 was excised from pCDNA3, together with the HA tag, by BamHI/XhoI digestion and cloned into the vector pHR-CMV-eGFP upstream of an internal ribosome entry site-driven enhanced green fluorescent protein gene (eGFP).
Production and measurement of lentivirus.
Lentivirus was produced by transient transfection of 293T cells with a weight ratio of 3 : 1 : 1 of vector to packaging to envelope plasmids using Fugene 6 (Roche) according to the manufacturer’s instructions. Control lentivirus was produced by co-transfection of the packaging and envelope plasmid together with the empty pHR-CMV-eGFP plasmid. For production of recombinant lentivirus expressing ORF20, the plasmid pHR-CMV-ORF20eGFP was used. Supernatants containing the lentivirus were collected at 48, 72 and 96 h post-transfection and clarified by low-speed centrifugation, and the lentivirus was collected by ultracentrifugation (25 000 r.p.m. in an SW28 rotor in a Beckman centrifuge). Virus pellets were resuspended in fresh culture medium and frozen at –80 °C. Lentivirus titres were measured by infection of 293T cells with a dilution factor of 4. Analysis of lentivirus-infected cells was done by detecting eGFP-positive cells by flow cytometry at 48 h post-infection (p.i.).
Immunofluorescence.
NIH3T3 and HeLa cells were infected with recombinant lentivirus containing the MHV68 ORF20 gene (pHR-CMV-ORF20eGFP) or the control ‘empty’ lentivirus (pHR-CMV-eGFP) at an m.o.i. of 10. At the time points indicated, cells seeded onto coverslips were washed in PBS and fixed with 4 % paraformaldehyde in PBS (20 min, room temperature). Fixed cells were permeabilized using PBS/0.1 % Triton-X 100 (20 min, room temperature). After washing with PBS, the fixed cells were blocked in PBS/5 % BSA/0.05 % Tween 20 (30 min, room temperature) prior to incubation with antibodies.
High-affinity anti-HA (Roche) and anti-phospho-histone H3 (Upstate Biotechnology) antibody were diluted in PBS/0.05 % Tween 20/1 % BSA and incubated with the specimen (1 h, room temperature). After three 5 min washes in PBS/0.05 % Tween 20, coverslips were incubated with Alexa Fluor anti-rat secondary antibody (Jackson ImmunoResearch) diluted in PBS/1 % BSA/0.05 % Tween 20 (1 h, room temperature). After washing with PBS/0.05 % Tween 20, coverslips were counterstained with the nuclear stain 4-6-diamino-2-phenylindole (DAPI) at 20 ng ml–1 in PBS (2 min, room temperature), mounted in Vectashield (Vector Laboratories) and observed under a fluorescence microscope.
Cell-cycle analysis.
NIH3T3 cells were infected with recombinant pHR-CMV-ORF20eGFP or control pHR-CMV-eGFP lentivirus at an m.o.i. of 10. At the indicated time points, cells were trypsinized (Gibco-BRL), washed once with PBS and fixed with 90 % ethanol overnight at 4 °C. After fixation, cells were washed once with PBS and resuspended in PBS/0.1 % Triton X-100 and incubated with 50U DNase-free RNase A (Calbiochem) (30 min, room temp). After incubation, cells were stained with propidium iodide (Sigma) (20 µg ml–1 in PBS, 15 min, room temperature) before analysis. Flow cytometry analysis was performed using a FACSCalibur (Becton Dickinson) and cell-cycle analysis was performed using CELLQUEST software.
TdT-mediated dUTP nick end labelling (TUNEL).
To determine the percentage of apoptosis, a FlowTACs kit (R&D Systems) was used as specified by the manufacturer. Briefly, at 72 h p.i., cells were trypsinized, washed with PBS and fixed in 3.7 % formaldehyde solution (10 min, room temperature). After fixation, cells were washed in PBS, permeabilized in Cytonin and incubated in labelling reaction mix (1 h, 37 °C). The reaction was terminated by adding ‘stop’ buffer, cells were centrifuged and visualization of the apoptotic cells was achieved by incubation with streptavidin–allophycocyanin (BD Biosciences Pharmingen) (15 min, room temperature, in the dark). Positive and negative controls were prepared using the same procedure and as provided by the supplier. Cells were washed in PBS and analysed using a FACSCalibur (Becton Dickinson) and data analysis was performed using CELLQUEST software.
Immunoblotting.
Cells cultured in 60 mm diameter Petri dishes were infected with different lentivirus constructs at an m.o.i. of 10 and harvested at various times p.i. The dishes were placed on ice and washed in PBS before scraping off the cells.
Cells were lysed in 50 µl lysis buffer containing 0.1 M potassium phosphate, 1 % Triton X-100, 2 mM EDTA, 1 mM dithiothreitol, 1 mM PMSF and a mixture of protease (Sigma) and phosphatase (Roche) inhibitors (30 min, 0 °C). Protein concentrations were determined by the Bradford assay (Bio-Rad). For SDS-PAGE, 50 µg protein was loaded per lane and the separated proteins were transferred to PVDF membrane (Bio-Rad) and blocked with 5 % non-fat milk (1 h, room temperature). Development of the blots was performed with a monoclonal antibody for Cdc2 and polyclonal antibodies for cyclin B1 (Santa Cruz Biotechnology), anti-phospho-Tyr-15 Cdc2 (Cell Signalling Technology) and anti-
-tubulin (Sigma-Aldrich). Horseradish peroxidase-conjugated secondary antibodies were purchased from Dako. Finally, the identified molecules were revealed with SuperSignal chemiluminescent reagents (Perbio Science) according to the manufacturer’s instructions.
In vitro kinase assays.
A histone H1 kinase assay was performed to measure the kinase activity of the Cdc2–cyclin B complex. NIH3T3 cells infected with lentivirus constructs at an m.o.i. of 10 were lysed at 48 h p.i. in a buffer from Cell Signalling Technology containing 1 mM 
-glycerophosphate, 1 mM sodium orthovanadate and a mix of protease inhibitors from Roche. Lysates containing 600 µg protein were incubated for 1 h at 4 °C in 300 µl lysis buffer with 5 µg anti-cyclin B1 antibody (Santa Cruz Biotechnology) and immune complexes were collected on 30 µl of protein G–Sepharose beads. The beads were washed three times with lysis buffer and once with kinase buffer containing 20 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 0.25 mM dithiothreitol, before being incubated for 10 min at 30 °C in 30 µl kinase buffer containing 2.5 µCi [
-32P]ATP (Amersham-Pharmacia), 10 µg histone H1 (Sigma) and 50 µM ATP. The reaction mixture was centrifuged and SDS sample buffer was immediately added to the supernatant prior to 12 % SDS-PAGE. The gel was dried and exposed for autoradiography for 1 h at –80 °C. The protein G beads were collected from the kinase reaction, washed three times in kinase buffer and denatured in 30 µl SDS sample buffer for separation by SDS-PAGE and subsequent transfer to PVDF membrane (Bio-Rad). The membrane was blocked with 5 % non-fat milk (1 h, room temperature), developed with a monoclonal antibody for Cdc2 (Santa Cruz Biotechnology) and a secondary horseradish peroxidase-conjugated antibody, and revealed with SuperSignal chemiluminescent reagents (Perbio Science) according to the manufacturer’s instructions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, the expressed protein migrated with a molecular mass of 28 kDa.
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). Significantly, mouse NIH3T3 cells infected with ORF20-recombinant lentivirus failed to divide and died after 3–5 days, showing the typical fragmented nuclear morphology of apoptosis. Prior to this, the cells became enlarged and flat, suggesting that ORF20 might interfere with cell-cycle progression.
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, second row). As molecules involved in the cell cycle are highly conserved in mammalian cells, we repeated the same experiment with human 293T and HeLa cells and obtained a similarly increased G2/M : G1 ratio in both cell types (Fig. 3 bottom row; this only shows the experiment in the HeLa cells). Infection with a control, non-recombinant lentivirus did not change the cell-cycle profile (Fig. 3, top and third rows).
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, top and middle rows). To confirm this observation, the cells were also examined by FACS analysis at 72 h p.i. for levels of lentivirus infection (eGFP expression) and apoptosis by the TUNEL procedure. The level of infection was 80–90 % and almost 50 % of recombinant lentivirus-infected NIH3T3 (Fig. 4b) and HeLa (data not shown) cells were apoptotic. There were no significant levels of apoptosis in control cells infected with the lentivirus containing the empty vector. Identical results were obtained with human (HeLa) cells infected with control and recombinant lentivirus (data not shown).
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Lentivirus-infected NIH3T3 cells expressing ORF20 were stained with anti-phospho-histone H3 at 48 and 72 h after infection with recombinant lentivirus. Only cells negative for the GFP marker of infection stained positively for phospho-histone H3, whereas lentivirus-infected cells did not express phospho-histone H3 (Fig. 5). This experiment demonstrated that cells infected with ORF20 arrest in G2 and enter apoptosis without entering mitosis.
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To test whether ORF20 was arresting the cell cycle in G2 and acting at the level of the cyclin complex, NIH3T3 cells were infected with lentivirus expressing ORF20 and cyclin B expression was determined by Western blot analysis with anti-cyclin B antibody. A clearly increased level of cyclin B was observed in recombinant lentivirus-infected cells from 48 to 60 h p.i., consistent with arrest at the G2/M phase of the cell cycle (Fig. 6). As the dephosphorylated form of Cdc2 is required for entry into mitosis, a similar experiment was performed, but the Western blots were developed with an antibody specific for the Tyr-15-phosphorylated form of Cdc2. The experiment revealed an increase in the phosphorylated (inactive) form of Cdc2 relative to the expression of total Cdc2 from 24 to 72 h after infection with recombinant lentivirus (Fig. 6). These changes were not observed when cells were transduced with the control, non-recombinant lentivirus. Similar results were obtained in human 293T cells (data not shown).
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To confirm the hypothesis that ORF20 induced G2 arrest by inactivation of the Cdc2–cyclin B complex, we compared the relative kinase activities of Cdc2–cyclin B in control and ORF20-expressing cells. Nocadazol-treated cells, which are blocked in mitosis, were used as a positive control. Lysates from NIH3T3 cells infected with recombinant lentivirus expressing ORF20 or with lentivirus control (eGFP) were prepared. Complexes of Cdc2–cyclin B were precipitated from cell lysates with protein G–Sepharose and an antibody to cyclin B, and their kinase activity was determined in a kinase assay using histone H1 as the substrate. The supernatants were examined for phosphorylated histone by SDS-PAGE and autoradiography (Fig. 7a) and the immunocomplexes bound to protein G were submitted to SDS-PAGE and Western blot analysis for anti-Cdc2 (Fig. 7b). Cells expressing ORF20 contained the higher-molecular-mass phosphorylated Cdc2 (inactive) complexed with cyclin B (Fig. 7b), which was almost completely inactive in the kinase assay (Fig. 7a). In contrast, in the nocadazol positive control, Cdc2 was present as the active non-phosphorylated form (Fig. 7b) and there was clear phosphorylation of the histone substrate in the kinase assay (Fig. 7a). The control, non-recombinant lentivirus (eGFP)-infected cell lysate contained both active and inactive forms of Cdc2, which catalysed a lower level of histone phosphorylation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Song et al., 2000; Sunil-Chandra et al., 1992a) and cell-cycle progression (Boldogh et al., 1990; Hobbs & DeLuca, 1999)
The key element in the regulation of the eukaryotic cell cycle is the periodic synthesis and destruction of cyclins, the proteins that associate and activate cyclin-dependent kinases. The related sequential activation and inactivation of cyclin-dependent kinases, through the periodic synthesis and destruction of cyclins, provide the basis of cell-cycle regulation. In addition to the formation of cyclin complexes, the phosphorylation state of cyclin-dependent kinases controls the activity of these kinases during the cell cycle. The key downstream target of G2 arrest is the mitosis-promoting kinase complex Cdc2–cyclin B. Entry into mitosis requires activation of Cdc2 following dephosphorylation of Tyr-15 and Thr-14. Various pathways converge at this point, shifting the balance towards entry into mitosis or arrest at G2 (Abraham, 2001; Castedo et al., 2002; Kastan & Bartek, 2004; Lukas & Bartek, 2004). In this work, we demonstrated that the non-essential ORF20 gene of the mouse herpesvirus MHV68 arrests cells in the G2 phase of the cell cycle (Fig. 3
) in both human and mouse cells, followed by induction of apoptosis (Fig. 4). Our observation that the ORF20 gene of MHV68 induces cell-cycle arrest in both human and mouse cells strongly suggests that this virus gene plays a fundamental role in herpesvirus strategy for host-cell modification and adaptation, and one that might be exploited for the rational understanding and control of these important human infectious agents.
The general mechanism of ORF20 focuses on the inactivation of the Cdc2–cyclin B complex through a block in the dephosphorylation-dependent activation of the Cdc2 molecule, as indeed occurs with the human papilloma virus (HPV) E2 protein (Fournier et al., 1999) and the human immunodeficiency virus (HIV) Vpr protein (He et al., 1995). Consequently, in cells transduced with ORF20, there is an increase in Cdc2 phosphorylated at the inhibitory site (Fig. 6). The exact sequence of events in the inhibition is still to be elucidated, but the localization of ORF20 to the nucleus (Fig. 2), as for its herpes simplex virus 1 (HSV-1) homologue (Pearson & Coen, 2002), suggests a primary influence on transcription. The HSV-1 homologue of ORF20, UL24, is not required for growth in cultured cells (Pearson & Coen, 2002), but, in mice, mutation of the HSV-1 UL24 gene impairs virus replication in tissue culture and in mouse eye (Jacobson et al., 1998). The fact that ORF20 is a virion-associated protein (Bortz et al., 2003) suggests that an early impact of the virus on cell-cycle progression may be a necessary aspect of the function of ORF20. However, the observation that ORF20 is a late protein (Ebrahimi et al., 2003) suggests a contributing requirement, either in cell-cycle control or in some other function. As mentioned above, several viral proteins induce G2 arrest. Although the mechanism of action of these viral proteins has not been elucidated fully, both the E2 gene from HPV and the Vpr gene from HIV promote G2 arrest by inactivation of Cdc2–cyclin B1 (Fournier et al., 1999; He et al., 1995). In the case of Vpr, one suggestion is to maximize viral production by delaying the death of infected cells and maintaining the host cell in a stage of the cell cycle (G2) in which transcription of the viral long-terminal repeat is optimal (Goh et al., 1998; Groschel & Bushman, 2005; Zhu et al., 2001).
Until now, as demonstrated here, cell-cycle arrest promoted by MHV68 or by any of its genes has not been described. Only recently, a viral D-type cyclin common to all gamma2-herpesvirus has been characterized in MHV68 (Hoge et al., 2000; Upton et al., 2005) This viral cyclin is not required for virus replication, either in vitro or in vivo, but is required for efficient reactivation from latency (Hoge et al., 2000). Like the MHV68 ORF20 gene, the precise biological role of this virus strategy for host manipulation is not known.
In conclusion, ORF20 of the mouse gammaherpesvirus MHV68 is a virion-associated protein with nuclear localization, which has evolved for host cell-cycle arrest at G2 through inactivation of the Cdc2–cyclin B complex. A more profound understanding of its mechanism and function provides a fascinating problem for further investigation. For example, whether the observed apoptosis induced by ORF20 is a default consequence of the G2 arrest or an independent mechanism will be addressed by deletion mutant analysis. A similar functional analysis of the homologous human herpesviruses gene UL24 is clearly a priority.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 22 September 2006;
accepted 17 January 2007.
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