Human cytomegalovirus (HCMV) infection causes a rapid induction of c-Fos and c-Human cytomegalovirus (HCMV) replication begins with the expression of the major immediate-early (MIE) gene products IE1 and IE2, which are multifunctional proteins mainly involved in regulating both viral and cellular gene expression (reviewed in reference 51). The MIE proteins are essential for the progression of the replication cycle and crucial determinants of the transition from latency to reactivation (62, 63). Hence, the regulation of their expression is a key point in controlling the outcome of the HCMV infectious programs.Transcription of the HCMV MIE genes is driven by a complex and potent promoter, the MIE promoter (MIEP), which comprises different functional units including a basal promoter, the enhancer region, and the modulator (23). The MIEP contains binding sites for a diverse set of signal-regulated stimulatory and inhibitory transcription factors, such as NF-B, ATF/CREB, activator protein 1 (AP-1), YY1, Sp1/ Sp3, and retinoic acid receptor (RAR)/retinoid X receptor (RXR), most of them densely packed in the enhancer region (48). In addition, viral tegument proteins and the MIE proteins themselves have also been shown to modulate MIEP activity. During latency, the MIEP is associated with markers of repressed heterochromatin, remaining silent (53, 59). Cellular differentiation and alterations in the levels of specific transcription factors by a variety of stimuli promote the activation of the MIEP and thereby the expression of downstream MIE genes. Consequently, MIEP activity is dependent on cell type, cellular differentiation stage, and the activity of specific signaling transduction pathways. Work with transgenic mice carrying a LacZ reporter under the control of the HCMV MIEP enhancer indicated that the expression of the MIEP is restricted to specific cell types in multiple organs, paralleling tissues normally infected by HCMV in the natural host (5,6,41). A number of studies in the last several years have addressed the relevance of different segments of the MIEP for MIE gene expression and viral replication (27,33,34,47,49). While the more distal component of the enhancer (spanning from Ϫ550 to Ϫ300 relative to the transcription start site [ϩ1] of the MIEP) has been shown only to partially contribute to viral replication at a low multiplicity of infection (MOI) (47), progressive deletions starting from the distal end of the proximal segment of the enhancer (spanning from Ϫ300 to Ϫ39) resulted in recombinant viruses that replicated slower and with
fWe have investigated the previously uncharacterized human cytomegalovirus (HCMV) UL1 open reading frame (ORF), a member of the rapidly evolving HCMV RL11 family. UL1 is HCMV specific; the absence of UL1 in chimpanzee cytomegalovirus (CCMV) and sequence analysis studies suggest that UL1 may have originated by the duplication of an ancestor gene from the RL11-TRL cluster (TRL11, TRL12, and TRL13). Sequence similarity searches against human immunoglobulin (Ig)-containing proteins revealed that HCMV pUL1 shows significant similarity to the cellular carcinoembryonic antigen-related (CEA) protein family N-terminal Ig domain, which is responsible for CEA ligand recognition. Northern blot analysis revealed that UL1 is transcribed during the late phase of the viral replication cycle in both fibroblast-adapted and endotheliotropic strains of HCMV. We characterized the protein encoded by hemagglutinin (HA)-tagged UL1 in the AD169-derived HB5 background. UL1 is expressed as a 224-amino-acid type I transmembrane glycoprotein which becomes detectable at 48 h postinfection. In infected human fibroblasts, pUL1 colocalized at the cytoplasmic site of virion assembly and secondary envelopment together with TGN-46, a marker for the trans-Golgi network, and viral structural proteins, including the envelope glycoprotein gB and the tegument phosphoprotein pp28. Furthermore, analyses of highly purified AD169 UL1-HA epitope-tagged virions revealed that pUL1 is a novel constituent of the HCMV envelope. Importantly, the deletion of UL1 in HCMV TB40/E resulted in reduced growth in a cell type-specific manner, suggesting that pUL1 may be implicated in regulating HCMV cell tropism.
Viral engagement with macrophages activates Toll-Like-Receptors (TLRs) and viruses must contend with the ensuing inflammatory responses to successfully complete their replication cycle. To date, known counter-strategies involve the use of viral-encoded proteins that often employ mimicry mechanisms to block or redirect the host response to benefit the virus. Whether viral regulatory DNA sequences provide an opportunistic strategy by which viral enhancer elements functionally mimic innate immune enhancers is unknown. Here we find that host innate immune genes and the prototypical viral enhancer of cytomegalovirus (CMV) have comparable expression kinetics, and positively respond to common TLR agonists. In macrophages but not fibroblasts we show that activation of NFκB at immediate-early times of infection is independent of virion-associated protein, M45. We find upon virus infection or transfection of viral genomic DNA the TLR-agonist treatment results in significant enhancement of the virus transcription-replication cycle. In macrophage time-course infection experiments we demonstrate that TLR-agonist stimulation of the viral enhancer and replication cycle is strictly delimited by a temporal gate with a determined half-maximal time for enhancer-activation of 6 h; after which TLR-activation blocks the viral transcription-replication cycle. By performing a systematic siRNA screen of 149 innate immune regulatory factors we identify not only anticipated anti-viral and pro-viral contributions but also new factors involved in the CMV transcription-replication cycle. We identify a central convergent NFκB-SP1-RXR-IRF axis downstream of TLR-signalling. Activation of the RXR component potentiated direct and indirect TLR-induced activation of CMV transcription-replication cycle; whereas chromatin binding experiments using wild-type and enhancer-deletion virus revealed IRF3 and 5 as new pro-viral host transcription factor interactions with the CMV enhancer in macrophages. In a series of pharmacologic, siRNA and genetic loss-of-function experiments we determined that signalling mediated by the TLR-adaptor protein MyD88 plays a vital role for governing the inflammatory activation of the CMV enhancer in macrophages. Downstream TLR-regulated transcription factor binding motif disruption for NFκB, AP1 and CREB/ATF in the CMV enhancer demonstrated the requirement of these inflammatory signal-regulated elements in driving viral gene expression and growth in cells as well as in primary infection of neonatal mice. Thus, this study shows that the prototypical CMV enhancer, in a restricted time-gated manner, co-opts through DNA regulatory mimicry elements, innate-immune transcription factors to drive viral expression and replication in the face of on-going pro-inflammatory antiviral responses in vitro and in vivo and; suggests an unexpected role for inflammation in promoting acute infection and has important future implications for regulating latency.
Major immediate-early transcriptional enhancers are genetic control elements that act, through docking with host transcription factors, as a decisive regulatory unit for efficient initiation of the productive virus cycle. Animal models are required for studying the function of enhancers paradigmatically in host organs. Here, we have sought to quantitatively assess the establishment, maintenance, and level of in vivo growth of enhancerless mutants of murine cytomegalovirus in comparison with those of an enhancer-bearing counterpart in models of the immunocompromised or immunologically immature host. Evidence is presented showing that enhancerless viruses are capable of forming restricted foci of infection but fail to grow exponentially.Transcriptional enhancers consist of modules of transcription factor binding sites and mediate gene desilencing and promoter activation in an orientation-independent manner and even from remote sites (4). Whereas the "rheostatic" model, also known as "rate" or "progressive-response" model, proposes that enhancers function by increasing the rate of transcription from a cognate gene, the "binary" model, also known as "on-or-off" or "probability" model, proposes that they function by raising the probability for a cognate gene being in a desilenced state (12). In either case, the result of enhancer action is an increased amount of transcripts. Major immediate-early (MIE) enhancers of herpesviruses enhance the transcription of MIE genes that encode transactivator proteins critically involved in the expression of viral early-phase (E) genes as well as host cell genes. Enhancers are therefore regarded as key regulators for initiating the productive cycle in acute infection as well as in the reactivation from latency. 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). 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).Whereas much is known from infected cell cultures, studies of MIE enhancer function in vivo are restricted to animal models. Accordingly, our knowledge of the in vivo role for MIE enhancers is still incomplete. Notably, orthologous enhancers of different CMV species can replace each other mor...
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