Cell Cycle-Independent Expression of Immediate-Early Gene 3 Results in G ₁ and G ₂ Arrest in Murine Cytomegalovirus-Infected Cells

Abstract

The infectious cycle of human cytomegalovirus (HCMV) is intricately linked to the host's cell cycle. Viral gene expression can be initiated only in G 0 /G 1 phase. Once expressed, the immediate-early gene product IE2 prevents cellular DNA synthesis, arresting infected cells with a G 1 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 G 1 . Unexpectedly, and in contrast to HCMV, MCMV can also block cells that have passed through S phase by arresting them in G 2 . Moreover, MCMV can also replicate in G 2 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 G 1 or G 2 . 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.Cytomegaloviruses (CMVs), the prototypical members of the betaherpesvirus subfamily, are ubiquitous, species-specific pathogens that typically establish an asymptomatic, lifelong infection in the immunocompetent host. In the immunocompromised host, CMV can reactivate and cause severe or even life-threatening disease. Consequently, human CMV (HCMV) infections are particularly hazardous in neonates as well as transplant recipients and AIDS patients.During their evolution, CMVs have evolved many ways to functionally interfere with their host's biology in order to ensure their replication and survival (43). In this respect, the analysis of cell cycle regulation by HCMV has been a constant focus of interest over recent years and by now has been studied to some extent mainly in lytically infected fibroblasts (2,12,16,29,55). These studies revealed a complex interplay between HCMV and the cell cycle state of the host cell. HCMV can infect cells throughout the cell cycle, but for unknown reasons viral de novo gene expression is initiated only in G 0 and early to mid-G 1 phases (17, 51). In later cell cycle phases, viral entry takes place, but viral gene expression is delayed until cells have divided and reentered the following G 1 phase (17, 51). There, or indeed also in G 0 cells, HCMV stimulates proproliferative cellular pathways by activating the S-phase-promoting cyclin E-Cdk2-Rb-E2F pathway (6,9,18,21,24,26,28,41,49,56,64,67,69) and the M-phase-promoting cyclin B1-Cdk1 complex (26,52,61,65,67). However, once infected cells reach the G 1 /S transition, the viral IE...

Human cytomegalovirus tegument protein pp150 acts as a cyclin A2–CDK-dependent sensor of the host cell cycle and differentiation state

“…Despite the presence of a pp150 homolog (M32), gene expression of murine CMV (MCMV) occurs both in a cell cycle and cyclin A2-independent manner (10,15). Aiming for evolutionary insight into HCMV's cell cycle dependency, we compared known pp150 homologs of mammalian CMVs.…”

“…It complements previous studies on the role of p53-p21-CDK signaling (9, 10) by adding a viral sensor molecule to this pathway. It explains why MCMV, used in the most common animal models of HCMV infection, was found to be less sensitive to varying cell cycle conditions and differentiation states (10,15,21) and it helps to understand the limited efficacy of HDAC inhibition or Daxx knockdown in desilencing IE gene expression in pluripotent cells (6).…”

The Canonical Immediate Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication but Mediates Transcriptional and Posttranscriptional Regulation of Viral Gene Products

“…Although wt MCMV infection results in G 1 and G 2 arrest and the ⌬ie3 virus has lost this ability, ectopically expressed pIE611 exhibits cell cycle arrest activity only in G 1 and not G 2 (29), exemplifying the occasion for reevaluation. Our findings open up the possibility to dissect the individual impact of each IE3 isoform for CMV pathogenesis and host cell interaction.…”

Inhibition of Human Cytomegalovirus Immediate-Early Gene Expression by Cyclin A2-Dependent Kinase Activity

“…Due to IE2-responsive promoter elements within the viral origin of replication (oriLyt), IE2 is also directly involved in the initiation of viral DNA synthesis (77). Therefore, IE2 has attracted considerable interest as an antiviral target (66, 74), and an IE2-specific antisense RNA is already in clinical use for the treatment of HCMV retinitis (26).Recently, we and others have shown that cells in the S/G 2 phase of the cell division cycle exhibit a broad block to HCMV IE gene expression (14,55,73). This block acts at the mRNA expression level and affects not only IE1 and IE2, but also other IE gene loci, like .…”