Synergism between the components of the bipartite major immediate-early transcriptional enhancer of murine cytomegalovirus does not accelerate virus replication in cell culture and host tissues

Abstract

Short CommunicationSynergism between the components of the bipartite major immediate-early transcriptional enhancer of murine cytomegalovirus does not accelerate virus replication in cell culture and host tissues Major immediate-early (MIE) transcriptional enhancers of cytomegaloviruses are key regulators that are regarded as determinants of virus replicative fitness and pathogenicity. The MIE locus of murine cytomegalovirus (mCMV) shows bidirectional gene-pair architecture, with a bipartite enhancer flanked by divergent core promoters. Here, we have constructed recombinant viruses mCMV-DEnh1 and mCMV-DEnh2 to study the impact of either enhancer component on bidirectional MIE gene transcription and on virus replication in cell culture and various host tissues that are relevant to CMV disease. The data revealed that the two unipartite enhancers can operate independently, but synergize in enhancing MIE gene expression early after infection. Kick-start transcription facilitated by the bipartite enhancer configuration, however, did not ultimately result in accelerated virus replication. We conclude that virus replication, once triggered, proceeds with a fixed speed and we propose that synergism between the components of the bipartite enhancer may rather increase the probability for transcription initiation.Transcriptional enhancers are defined as cis-acting genetic elements that are composed of modules of transcription factor-binding sites to increase transcription in an orientation-independent fashion and even from a distance (Blackwood & Kadonaga, 1998;Fiering et al., 2000). The major immediate-early (MIE) locus of murine cytomegalovirus (mCMV) Dorsch-Häsler et al., 1985;Keil et al., 1987a;Maul & Negorev, 2008), like that of its human cytomegalovirus (hCMV) analogue (Boshart et al., 1985;Meier & Stinski, 2006;Stinski & Isomura, 2008), is a key regulatory region decisive for the initiation of the productive virus cycle in acute infection and in reactivation from latency. In both hCMV and mCMV, MIE locus activity is associated with gene desilencing by chromatin remodelling (Bain et al., 2006; Liu et al., 2008;Murphy et al., 2002).As discussed previously (Chatellard et al., 2007;Reddehase et al., 2008;Simon et al., 2007), the mCMV MIE locus shows a gene arrangement that is reminiscent of a bidirectional gene pair, a gene architecture frequently used in mammalian genomes for coordinated parallel or alternate expression of genes involved in vital functions such as DNA repair (Adachi & Lieber, 2002;Trinklein et al., 2004). By definition, a bidirectional gene pair consists of two divergently transcribed neighbouring genes arranged head to head on opposite strands of the DNA and regulated by a shared bidirectional promoter. Alternatively, bidirectionality can be achieved by placing two core promoters divergently on both upstream and downstream sides of a duplicated enhancer, a construct used in gene technology for enhanced and coordinated expression of transgenes (Li et al., 2004).Notably, such a promoter-enhancer-enh...

Reversible Inhibition of Murine Cytomegalovirus Replication by Gamma Interferon (IFN-γ) in Primary Macrophages Involves a Primed Type I IFN-Signaling Subnetwork for Full Establishment of an Immediate-Early Antiviral State

“…Total cellular RNA was isolated at 6 h postinfection (RNeasy minikit; Qiagen, Hilden, Germany), quality controlled, and transcribed into cDNA using an anchored poly(T) primer (Transcriptor first-strand cDNA synthesis kit; Roche Diagnostics, Burgess Hill, United Kingdom). DNA templates were diluted 1:10, and changes in viral gene expression levels were analyzed by relative quantitative real-time PCR using TaqMan primers and probe combinations as described elsewhere (43,51). Ready-to-use probe/primer sets were used to analyze tumor necrosis factor alpha (TNF-␣) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript levels (TaqMan gene expression assay; Applied Biosystems, Carlsbad, CA) as described elsewhere (44), and PCRs for detecting viral genes were carried out using HotStar Taq (Qiagen) as recommended by the manufacturer, except for increasing the MgCl 2 concentration to 2.5 mM.…”

Enhancerless Cytomegalovirus Is Capable of Establishing a Low-Level Maintenance Infection in Severely Immunodeficient Host Tissues but Fails in Exponential Growth

“…Thus, individual cells infected with enhancerless virus can show an almost WT phenotype with regard to IE1 expression and function, at least in the first round of infection, but appear to have a deficiency in replicative capacity and spread. In this context, it is worth recalling that mCMV⌬Enh.Luc virions were found to be poorly released into the cell culture supernatant (24), suggesting that focus formation may depend on cell-to-cell spread. Accordingly, we wondered if infected host tissue cells with such a phenotype might be founders of a progressive in vivo infection.…”

“…(Fig. 1B), consistent with reduced viral DNA replication and release of infectivity (24). Expression and functional activity of protein IE1 was studied on a single-cell level by confocal laser scanning microscopy (CLSM) correlating nuclear IE1 fluorescence intensity and IE1's capacity to dissociate repressive nuclear domains 10 (ND10), as described and illustrated with images in previous reports (14,39,44,46) (Fig.…”

“…IE3/M122, corresponding to IE2/UL122 of human CMV (hCMV), has been identified as the essential transactivator for E gene expression (30) and thus for viral replication (1). The two enhancer elements can act independently if engineered in isolation in viruses mCMV-⌬Enh1 or mCMV-⌬Enh2, and act synergistically after infection with wild-type (WT) virus (24).…”

“…The current knowledge of MIE enhancers of cytomegaloviruses (CMVs), members of the beta subfamily of the herpesviruses, and of the role of the cognate MIE proteins in the viral replication cycle have been comprehensively reviewed recently (3,6,26,27,28,36,42,43). In the specific case of murine CMV (mCMV), the MIE locus consists of a bidirectional gene pair with a promoter-enhancer-enhancer-promoter element (8,11,24,34,40) flanked to the left and to the right by transcription unit ie1-ie3 and gene ie2 that are transcribed in opposite directions and encode the transactivator proteins IE1 and IE3 as well as IE2, respectively (7,21,22,30,31). IE3/M122, corresponding to IE2/UL122 of human CMV (hCMV), has been identified as the essential transactivator for E gene expression (30) and thus for viral replication (1).…”