Human cytomegalovirus IE1-72 activates ataxia telangiectasia mutated kinase and a p53/p21-mediated growth arrest response

Human cytomegalovirus (HCMV) encodes several proteins that can modulate components of the cell cycle machinery. The UL123 gene product, IE1-72, binds the Rb-related, p107 protein and relieves its repression of E2F-responsive promoters; however, it is unable to induce quiescent cells to enter S phase in wild-type (p53(+/+)) cells. IE1-72 also induces p53 accumulation through an unknown mechanism. We present here evidence suggesting that IE1-72 may activate the p53 pathway by increasing the levels of p19(Arf) and by inducing the phosphorylation of p53 at Ser15. Phosphorylation of this residue by IE1-72 expression alone or HCMV infection is found to be dependent on the ataxia-telangiectasia mutated kinase. IE2-86 expression leads to p53 phosphorylation and may contribute to this phenotype in HCMV-infected cells. We also found that IE1-72 promotes p53 nuclear accumulation by abrogating p53 nuclear shuttling. These events result in the stimulation of p53 activity, leading to a p53- and p21-dependent inhibition of cell cycle progression from G(1) to S phase in cells transiently expressing IE1-72. Thus, like many of the small DNA tumor viruses, the first protein expressed upon HCMV infection activates a p53 response by the host cell.     

Inhibition of cell division by the human cytomegalovirus IE86 protein: role of the p53 pathway or cyclin-dependent kinase 1/cyclin B1

The human cytomegalovirus (HCMV) IE86 protein induces the human fibroblast cell cycle from G(0)/G(1) to G(1)/S, where cell cycle progression stops. Cells with a wild-type, mutated, or null p53 or cells with null p21 protein were transduced with replication-deficient adenoviruses expressing HCMV IE86 protein or cellular p53 or p21. Even though S-phase genes were activated in a p53 wild-type cell, IE86 protein also induced phospho-Ser(15) p53 and p21 independent of p14ARF but dependent on ATM kinase. These cells did not enter the S phase. In human p53 mutant, p53 null, or p21 null cells, IE86 protein did not up-regulate p21, cellular DNA synthesis was not inhibited, but cell division was inhibited. Cells accumulated in the G(2)/M phase, and there was increased cyclin-dependent kinase 1/cyclin B1 activity. Although the HCMV IE86 protein increases cellular E2F activity, it also blocks cell division in both p53(+/+) and p53(-/-) cells.     

Effect of Inducible Expressed Human Cytomegalovirus Immediate Early 86 Protein on Cell Apoptosis

Human cytomegalovirus is a common human pathogen that can cause life-threatening disease under certain conditions. During infection of host cells, the virus expresses regulatory proteins such as IE72 and IE86 that are important for viral propagation. IE86 plays a critical role in the modulation of viral replication as well as host cell cycle control and apoptosis. In this study, a Tet-On system was used to quantify the effect of IE86 on apoptosis and p53 expression. Our results indicate that IE86 inhibits tumor necrosis factor (TNF)-α induced apoptosis and that the anti-apoptotic activity of this viral protein correlates with its expression levels. In addition, IE86 did not alter the mRNA level of p53. The system developed should provide a method for functional analysis of human cytomegalovirus (HCMV) IE86 protein.     

The human cytomegalovirus IE86 protein can block cell cycle progression after inducing transition into the S phase of permissive cells

Human cytomegalovirus (HCMV) infection of permissive cells has been reported to induce a cell cycle halt. One or more viral proteins may be involved in halting progression at different stages of the cell cycle. We investigated how HCMV infection, and specifically IE86 protein expression, affects the cell cycles of permissive and nonpermissive cells. We used a recombinant virus that expresses the green fluorescent protein (GFP) to determine the effects of HCMV on the cell cycle of permissive cells. Fluorescence by GFP allowed us to select for only productively infected cells. Replication-defective adenovirus vectors expressing the IE72 or IE86 protein were also used to efficiently transduce 95% or more of the cells. The adenovirus-expressed IE86 protein was determined to be functional by demonstrating negative autoregulation of the major immediate-early promoter and activation of an early viral promoter in the context of the viral genome. To eliminate adenovirus protein effects, plasmids expressing GFP for fluorescent selection of only transfected cells and wild-type IE86 protein or a mutant IE86 protein were tested in permissive and nonpermissive cells. HCMV infection induced the entry of U373 cells into the S phase. All permissive cells infected with HCMV were blocked in cell cycle progression and could not divide. After either transduction or transfection and IE86 protein expression, the number of all permissive or nonpermissive cell types in the S phase increased significantly, but the cells could no longer divide. The IE72 protein did not have a significant effect on the S phase. Since IE86 protein inhibits cell cycle progression, the IE2 gene in a human fibroblast IE86 protein-expressing cell line was sequenced. The IE86 protein in these retrovirus-transduced cells has mutations in a critical region of the viral protein. The locations of the mutations and the function of the IE86 protein in controlling cell cycle progression are discussed.     

The Cellular Protein MCM3AP Is Required for Inhibition of Cellular DNA Synthesis by the IE86 Protein of Human Cytomegalovirus

Like all DNA viruses, human cytomegalovirus (HCMV) infection is known to result in profound effects on host cell cycle. Infection of fibroblasts with HCMV is known to induce an advance in cell cycle through the G₀-G₁ phase and then a subsequent arrest of cell cycle in early S-phase, presumably resulting in a cellular environment optimum for high levels of viral DNA replication whilst precluding replication of cellular DNA. Although the exact mechanisms used to arrest cell cycle by HCMV are unclear, they likely involve a number of viral gene products and evidence points to the ability of the virus to prevent licensing of cellular DNA synthesis. One viral protein known to profoundly alter cell cycle is the viral immediate early 86 (IE86) protein - an established function of which is to initially drive cells into early S phase but then inhibit cellular DNA synthesis. Here we show that, although IE86 interacts with the cellular licensing factor Cdt1, it does not inhibit licensing of cellular origins. Instead, IE86-mediated inhibition of cellular DNA synthesis requires mini-chromosome-maintenance 3 (MCM3) associated protein (MCM3AP), which can cause subsequent inhibition of initiation of cellular DNA synthesis in a licensing-independent manner.     

Cell cycle arrest by human cytomegalovirus 86-kDa IE2 protein resembles premature senescence

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display beta-galactosidase (beta-Gal) activity at neutral pH (senescence-associated beta-Gal [SA-beta-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16(INK4a), a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21(CIP1/WAF). Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (alpha) genes are sufficient for the induction of SA-beta-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16(INK4a) (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-beta-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16(INK4a) and p53 pathways.     

Alternative splicing of the human cytomegalovirus major immediate-early genes affects infectious-virus replication and control of cellular cyclin-dependent kinase

The major immediate-early (MIE) gene locus of human cytomegalovirus (HCMV) is the master switch that determines the outcomes of both lytic and latent infections. Here, we provide evidence that alteration in the splicing of HCMV (Towne strain) MIE genes affects infectious-virus replication, movement through the cell cycle, and cyclin-dependent kinase activity. Mutation of a conserved 24-nucleotide region in MIE exon 4 increased the abundance of IE1-p38 mRNA and decreased the abundance of IE1-p72 and IE2-p86 mRNAs. An increase in IE1-p38 protein was accompanied by a slight decrease in IE1-p72 protein and a significant decrease in IE2-p86 protein. The mutant virus had growth defects, which could not be complemented by wild-type IE1-p72 protein in trans. The phenotype of the mutant virus could not be explained by an increase in IE1-p38 protein, but prevention of the alternate splice returned the recombinant virus to the wild-type phenotype. The lower levels of IE1-p72 and IE2-p86 proteins correlated with a delay in early and late viral gene expression and movement into the S phase of the cell cycle. Mutant virus-infected cells had significantly higher levels of cdk-1 expression and enzymatic activity than cells infected with wild-type virus. The mutant virus induced a round-cell phenotype that accumulated in the G(2)/M compartment of the cell cycle with condensation and fragmentation of the chromatin. An inhibitor of viral DNA synthesis increased the round-cell phenotype. The round cells were characteristic of an abortive viral infection.     

HCMV IE86对恶性胶质瘤细胞凋亡及p53表达活性的影响

人巨细胞病毒(human cytomegalovirus,HCMV)能诱导肿瘤细胞恶性转化且抑制肿瘤细胞凋亡,但HCMV编码的主要即刻早期调控蛋白IE86在这一过程中是否发挥关键作用仍然未知。为探究IE86对基因修饰荷胶质瘤小鼠p53表达水平及恶性胶质瘤细胞凋亡情况的影响,本研究通过PCR技术鉴定基因修饰小鼠IE86表达情况;实时定量PCR技术检测IE86和p53mRNA表达水平变化;免疫组织化学方法检测p53和p21蛋白的表达水平;TUNEL检测肿瘤组织细胞凋亡情况。结果显示,成功构建了IE86基因修饰小鼠模型;与IE86阴性组相比IE86阳性组p53表达水平上升(P0.05),但p21表达水平下降(P0.05);IE86阳性组细胞抗凋亡能力增强(P0.05)。以上结果表明,在基因修饰的小鼠中IE86持续表达但p53转录活性的指示标志p21下调,且IE86可提高恶性胶质瘤细胞抗凋亡能力。     

Exon 3 of the human cytomegalovirus major immediate-early region is required for efficient viral gene expression and for cellular cyclin modulation

The human cytomegalovirus (HCMV) major immediate-early (IE) proteins share an 85-amino-acid N-terminal domain specified by exons 2 and 3 of the major IE region, UL122-123. We have constructed IE Delta30-77, a recombinant virus that lacks the majority of IE exon 3 and consequently expresses smaller forms of both IE1 72- and IE2 86-kDa proteins. The mutant virus is viable but growth impaired at both high and low multiplicities of infection and exhibits a kinetic defect that is not rescued by growth in fibroblasts expressing IE1 72-kDa protein. The kinetics of mutant IE2 protein accumulation in IE Delta30-77 virus-infected cells are approximately normal compared to wild-type virus-infected cells, but the IE Delta30-77 virus is delayed in expression of early viral genes, including UL112-113 and UL44, and does not sustain expression of mutant IE1 protein as the infection progresses. Additionally, cells infected with IE Delta30-77 exhibit altered expression of cellular proteins compared to wild-type HCMV-infected cells. PML is not dispersed but is retained at ND10 sites following infection with IE Delta30-77 mutant virus. While the deletion mutant retains the ability to mediate the stabilization of cyclin B1, cdc6, and geminin in infected cells, its capacity to upregulate the expression of cyclin E has been reduced. These data indicate that the activity of one or both of the HCMV major IE proteins is required in vivo for the modulation of cell cycle proteins observed in cells infected with wild-type HCMV.     

人巨细胞病毒即刻早期蛋白IE2在Tet-On系统调控下的表达及其抗凋亡作用

即刻早期蛋白IE2是人巨细胞病毒(HCMV)感染后最先表达的蛋白质,具有调节细胞周期和抑制细胞凋亡的作用。但IE2蛋白的表达水平与其抗凋亡活性之间的关系尚不清楚。本实验通过建立Tet-On系统调控下表达HCMVIE2蛋白的细胞株,在不同诱导条件下检测IE2蛋白对细胞凋亡和p53表达的影响。结果发现IE2蛋白能抑制TNF-α诱导的细胞凋亡,其抑制效应与IE2蛋白的表达量相关,而原位杂交结果显示IE2蛋白不影响p53的表达,提示IE2蛋白可能通过多种途径抑制细胞凋亡。     

SUMO-1 modification of human cytomegalovirus IE1/IE72

Human cytomegalovirus (HCMV) immediate-early protein IE1/IE72 is involved in undermining many cellular processes including cell cycle regulation, apoptosis, nuclear architecture, and gene expression. The multifunctional nature of IE72 suggests that posttranslational modifications may modulate its activities. IE72 is a phosphoprotein and has intrinsic kinase activity (S. Pajovic, E. L. Wong, A. R. Black, and J. C. Azizkhan, Mol. Cell. Biol. 17:6459-6464, 1997). We now demonstrate that IE72 is covalently conjugated to the small ubiquitin-like modifier (SUMO-1). SUMO-1 is an 11.5-kDa protein that is conjugated to multiple proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. A covalently modified protein migrating at approximately 92 kDa, which is stabilized by a SUMO-1 hydrolase inhibitor, is revealed by Western blotting with anti-IE72 of lysates from cells infected with HCMV or cells expressing IE72. SUMO modification of IE72 was confirmed by immunoprecipitation with anti-IE72 and anti-SUMO-1 followed by Western blotting with anti-SUMO-1 and anti-IE72, respectively. Lysine 450 is within a sumoylation consensus site (I,V,L)KXE; changing lysine 450 to arginine by point mutation abolishes SUMO-1 modification of IE72. Inhibition of protein phosphatase 1 and 2A, which increases the phosphorylation of IE72, suppresses the formation of SUMO-1-IE72 conjugates. Both wild-type IE72 and IE72(K450R) localize to nuclear PML oncogenic domains and disrupt them. Studies of protein stability, transactivation, and complementation of IE72-deficient HCMV (CR208) have revealed no significant differences between wild-type IE72 and IE72(K450R).     

Cell cycle-independent expression of immediate-early gene 3 results in G1 and G2 arrest in murine cytomegalovirus-infected cells

The infectious cycle of human cytomegalovirus (HCMV) is intricately linked to the host's cell cycle. Viral gene expression can be initiated only in G₀/G₁ phase. Once expressed, the immediate-early gene product IE2 prevents cellular DNA synthesis, arresting infected cells with a G₁ DNA content. This function is required for efficient viral replication in vitro. A prerequisite for addressing its in vivo relevance is the characterization of cell cycle-regulatory activities of CMV species for which animal models have been established. Here, we show that murine CMV (MCMV), like HCMV, has a strong antiproliferative capacity and arrests cells in G₁. Unexpectedly, and in contrast to HCMV, MCMV can also block cells that have passed through S phase by arresting them in G₂. Moreover, MCMV can also replicate in G₂ cells. This is made possible by the cell cycle-independent expression of MCMV immediate-early genes. Transfection experiments show that of several MCMV candidate genes, only immediate-early gene 3 (ie3), the homologue of HCMV IE2, exhibits cell cycle arrest activity. Accordingly, an MCMV ie3 deletion mutant has lost the ability to arrest cells in either G₁ or G₂. Thus, despite interspecies variations in the cell cycle dependence of viral gene expression, the central theme of HCMV IE2-induced cell cycle arrest is conserved in the murine counterpart, raising the possibility of studying its physiological relevance at the level of the whole organism.