Type I Interferon Released by Myeloid Dendritic Cells Reversibly Impairs Cytomegalovirus Replication by Inhibiting Immediate Early Gene Expression.

UNLABELLED: Cytomegalovirus (CMV) is a ubiquitous beta-herpesvirus whose reactivation from latency is a major cause of morbidity and mortality in immunocompromised hosts. Mouse CMV (MCMV) is a well-established model virus to study virus-host interactions. We showed in this study that the CD8-independent antiviral function of myeloid dendritic cells (mDC) is biologically relevant for the inhibition of MCMV replication in vivo and in vitro. In vivo ablation of CD11c(+) DC resulted in higher viral titers and increased susceptibility to MCMV infection in the first 3 days postinfection. We developed in vitro coculture systems in which we cocultivated MCMV-infected endothelial cells or fibroblasts with T cell subsets and/or dendritic cells. While CD8 T cells failed to control MCMV replication, bone marrow-derived mDC reduced viral titers by a factor of up to 10,000. Contact of mDC with the infected endothelial cells was crucial for their antiviral activity. Soluble factors secreted by the mDC blocked MCMV replication at the level of immediate early (IE) gene expression, yet the viral lytic cycle reinitiated once the mDC were removed from the cells. On the other hand, the mDC did not impair MCMV replication in cells deficient for the interferon (IFN) alpha/beta receptor (IFNAR), arguing that type I interferons were critical for viral control by mDC. In light of our recent observation that type I IFN is sufficient for the induction of latency immediately upon infection, our results imply that IFN secreted by mDC may play an important role in the establishment of CMV latency. IMPORTANCE: Numerous studies have focused on the infection of DC with cytomegaloviruses and on the establishment of latency within them. However, almost all of these studies have relied on the infection of DC monocultures in vitro, whereas DC are just one among many cell types present in an infection site in vivo. To mimic this aspect of the in vivo situation, we cocultured DC with infected endothelial cells or fibroblasts. Our data suggest that direct contact with virus-infected endothelial cells activates CD11c(+) DC, which leads to reversible suppression of MCMV replication at the level of IE gene expression by a mechanism that depends on type I IFN. The effect matches the formal definition of viral latency. Therefore, our data argue that the interplay of dendritic cells and infected neighboring cells might play an important role in the establishment of viral latency.

Reversible silencing of cytomegalovirus genomes by type I interferon governs virus latency.

Herpesviruses establish a lifelong latent infection posing the risk for virus reactivation and disease. In cytomegalovirus infection, expression of the major immediate early (IE) genes is a critical checkpoint, driving the lytic replication cycle upon primary infection or reactivation from latency. While it is known that type I interferon (IFN) limits lytic CMV replication, its role in latency and reactivation has not been explored. In the model of mouse CMV infection, we show here that IFNß blocks mouse CMV replication at the level of IE transcription in IFN-responding endothelial cells and fibroblasts. The IFN-mediated inhibition of IE genes was entirely reversible, arguing that the IFN-effect may be consistent with viral latency. Importantly, the response to IFNß is stochastic, and MCMV IE transcription and replication were repressed only in IFN-responsive cells, while the IFN-unresponsive cells remained permissive for lytic MCMV infection. IFN blocked the viral lytic replication cycle by upregulating the nuclear domain 10 (ND10) components, PML, Sp100 and Daxx, and their knockdown by shRNA rescued viral replication in the presence of IFNß. Finally, IFNß prevented MCMV reactivation from endothelial cells derived from latently infected mice, validating our results in a biologically relevant setting. Therefore, our data do not only define for the first time the molecular mechanism of IFN-mediated control of CMV infection, but also indicate that the reversible inhibition of the virus lytic cycle by IFNß is consistent with the establishment of CMV latency.

M27 expressed by cytomegalovirus counteracts effective type I interferon induction of myeloid cells but not of plasmacytoid dendritic cells.

UNLABELLED: In healthy individuals, the functional immune system effectively confines human cytomegalovirus (CMV) replication, while viral immune evasion and persistence preclude sterile immunity. Mouse CMV (MCMV) is a well-established model to study the delicate CMV-host balance. Effective control of MCMV infection depends on the induction of protective type I interferon (IFN-I) responses. Nevertheless, it is unclear whether in professional antigen-presenting cell subsets MCMV-encoded evasins inhibit the induction of IFN-I responses. Upon MCMV treatment, enhanced expression of MCMV immediate-early and early proteins was detected in bone marrow cultures of macrophages and myeloid dendritic cells compared with plasmacytoid dendritic cell cultures, whereas plasmacytoid dendritic cells mounted more vigorous IFN-I responses. Experiments with Toll-like receptor (TLR)- and/or RIG-I like helicase (RLH)-deficient cell subsets revealed that upon MCMV treatment of myeloid cells, IFN-I responses were triggered independently of TLR and RLH signaling, whereas in plasmacytoid dendritic cells, IFN-I induction was strictly TLR dependent. Macrophages and myeloid dendritic cells treated with either UV-inactivated MCMV or live MCMV that lacked the STAT2 antagonist M27 mounted significantly higher IFN-I responses than cells treated with live wild-type MCMV. In contrast, plasmacytoid dendritic cells responded similarly to UV-inactivated and live MCMV. These experiments illustrated that M27 not only inhibited IFN-I-mediated receptor signaling, but also evaded the induction of IFN responses in myeloid dendritic cells. Furthermore, we found that additional MCMV-encoded evasins were needed to efficiently shut off IFN-I responses of macrophages, but not of myeloid dendritic cells, thus further elucidating the subtle adjustment of the host-pathogen balance. IMPORTANCE: MCMV may induce IFN-I responses in fibroblasts and epithelial cells, as well as in antigen-presenting cell subsets. We focused on the analysis of IFN-I responses of antigen-presenting cell subsets, including plasmacytoid dendritic cells, myeloid dendritic cells, and macrophages, which are all triggered by MCMV to mount IFN-I responses. Interestingly, myeloid dendritic cells and macrophages, but not plasmacytoid dendritic cells, are readily MCMV infected and support viral gene expression. As expected from previous studies, plasmacytoid dendritic cells sense MCMV Toll-like receptor 9 (TLR9) dependently, whereas in myeloid cells, IFN-I induction is entirely TLR and RLH independent. MCMV-encoded M27 does not impair the IFN-I induction of plasmacytoid dendritic cells, while in myeloid dendritic cells, it reduces IFN-I responses. In macrophages, M27 plus other, not yet identified evasins profoundly inhibit the induction of IFN-I responses. Collectively, these results illustrate that MCMV has evolved diverse mechanisms to differentially modulate IFN-I responses in single immune cell subsets.

Cytomegalovirus immune evasion of myeloid lineage cells.

Cytomegalovirus (CMV) evades the immune system in many different ways, allowing the virus to grow and its progeny to spread in the face of an adverse environment. Mounting evidence about the antiviral role of myeloid immune cells has prompted the research of CMV immune evasion mechanisms targeting these cells. Several cells of the myeloid lineage, such as monocytes, dendritic cells and macrophages, play a role in viral control, but are also permissive for CMV and are naturally infected by it. Therefore, CMV evasion of myeloid cells involves mechanisms that qualitatively differ from the evasion of non-CMV-permissive immune cells of the lymphoid lineage. The evasion of myeloid cells includes effects in cis, where the virus modulates the immune signaling pathways within the infected myeloid cell, and those in trans, where the virus affects somatic cells targeted by cytokines released from myeloid cells. This review presents an overview of CMV strategies to modulate and evade the antiviral activity of myeloid cells in cis and in trans.

A new reporter mouse cytomegalovirus reveals maintained immediate-early gene expression but poor virus replication in cycling liver sinusoidal endothelial cells.

BACKGROUND: The MCMV major immediate early promoter/enhancer (MIEP) is a bidirectional promoter that drives the expression of the three immediate early viral genes, namely ie1, ie2 and ie3. The regulation of their expression is intensively studied, but still incompletely understood. METHODS: We constructed a reporter MCMV, (MCMV-MIEPr) expressing YFP and tdTomato under the control of the MIEP as proxies of ie1 and ie2, respectively. Moreover, we generated a liver sinusoidal endothelial cell line (LSEC-uniLT) where cycling is dependent on doxycycline. We used these novel tools to study the kinetics of MIEP-driven gene expression in the context of infection and at the single cell level by flow cytometry and by live imaging of proliferating and G0-arrested cells. RESULTS: MCMV replicated to higher titers in G0-arrested LSEC, and cycling cells showed less cytopathic effect or YFP and tdTomato expression at 5 days post infection. In the first 24 h post infection, however, there was no difference in MIEP activity in cycling or G0-arrested cells, although we could observe different profiles of MIEP gene expression in different cell types, like LSECs, fibroblasts or macrophages. We monitored infected LSEC-uniLT in G0 by time lapse microscopy over five days and noticed that most cells survived infection for at least 96 h, arguing that quick lysis of infected cells could not account for the spread of the virus. Interestingly, we noticed a strong correlation between the ratio of median YFP and tdTomato expression and length of survival of infected cells. CONCLUSION: By means of our newly developed genetic tools, we showed that the expression pattern of MCMV IE1 and IE2 genes differs between macrophages, endothelial cells and fibroblasts. Substantial and cell-cycle independent differences in the ie1 and ie2 transcription could also be observed within individual cells of the same population, and marked ie2 gene expression was associated with longer survival of the infected cells.