Mouse Cytomegalovirus Research Articles

Molecular dissection of plasmacytoid dendritic cell activation in vivo during a viral infection

Plasmacytoid dendritic cells (pDC) are the major source of type I interferons (IFN‐I) during viral infections, in response to triggering of endosomal Toll‐like receptors (TLRs) 7 or 9 by viral single‐stranded RNA or unmethylated CpG DNA, respectively. Synthetic ligands have been used to disentangle the underlying signaling pathways. The adaptor protein AP3 is necessary to transport molecular complexes of TLRs, synthetic CpG DNA, and MyD88 into endosomal compartments allowing interferon regulatory factor 7 (IRF7) recruitment whose phosphorylation then initiates IFN‐I production. High basal expression of IRF7 by pDC and its further enhancement by positive IFN‐I feedback signaling appear to be necessary for robust cytokine production. In contrast, we show here that in vivo during mouse cytomegalovirus (MCMV) infection pDC produce high amounts of IFN‐I downstream of the TLR9‐to‐MyD88‐to‐IRF7 signaling pathway without requiring IFN‐I positive feedback, high IRF7 expression, or AP3‐driven endosomal routing of TLRs. Hence, the current model of the molecular requirements for professional IFN‐I production by pDC, established by using synthetic TLR ligands, does not strictly apply to a physiological viral infection.

Endogenous glucocorticoids control host resistance to viral infection through the tissue-specific regulation of PD-1 expression on NK cells.

Controlling the balance between immunity and immunopathology is crucial for host resistance to pathogens. After infection, activation of the hypothalamic–pituitary–adrenal (HPA) axis leads to the production of glucocorticoids. However, the pleiotropic effects of these steroid hormones make it difficult to delineate their precise role(s) in vivo. Here we found that the regulation of natural killer (NK) cell function by the glucocorticoid receptor (GR) was required for host survival to mouse cytomegalovirus infection. Mechanistically, endogenous glucocorticoids produced shortly after infection induced the selective and tissue-specific expression of the checkpoint molecule PD-1 on NK cells. This glucocorticoid–PD-1 pathway limited the production of the cytokine interferon-γ (IFN-γ) by splenic NK cells, which prevented lethal immunopathology. Importantly, this regulation did not compromise viral clearance. Thus, the fine-tuning of NK cell functions by the HPA axis preserved tissue integrity without impairing pathogen elimination, which reveals a novel aspect of neuroimmune regulation.

Non-redundant ISGF3 Components Promote NK Cell Survival in an Auto-regulatory Manner during Viral Infection

SUMMARYNatural killer (NK) cells are innate lymphocytes that possess adaptive features, including antigen-specific clonal expansion and long-lived memory responses. Although previous work demonstrated that type I interferon (IFN) signaling is crucial for NK cell expansion and memory cell formation following mouse cytomegalovirus (MCMV) infection, the global transcriptional mechanisms underlying type I IFN-mediated responses remained to be determined. Here, we demonstrate that among the suite of transcripts induced in activated NK cells, IFN-α is necessary and sufficient to promote expression of its downstream transcription factors STAT1, STAT2, and IRF9, via an auto-regulatory, feedforward loop. Similar to STAT1 deficiency, we show that STAT2- or IRF9-deficient NK cells are defective in their ability to expand following MCMV infection, in part because of diminished survival rather than an inability to proliferate. Thus, our findings demonstrate that individual ISGF3 components are crucial cell-autonomous and non-redundant regulators of the NK cell response to viral infection.

IL-27 regulates the number, function and cytotoxic program of antiviral CD4 T cells and promotes cytomegalovirus persistence.

The role of IL-27 in antiviral immunity is still incompletely understood, especially in the context of chronic viruses that induce a unique environment in their infected host. Cytomegalovirus (CMV) establishes a persistent, tissue localized infection followed by lifelong latency. CMV infects the majority of people and although asymptomatic in healthy individuals, can cause serious disease or death in those with naïve or compromised immune systems. Therefore, there is an urgent need to develop a protective CMV vaccine for people at-risk and identifying key regulators of the protective immune response towards CMV will be crucial. Here we studied mouse CMV (MCMV) in IL-27 receptor deficient animals (Il27ra-/-) to assess the role of IL-27 in regulating CMV immunity. We found that IL-27 enhanced the number of antiviral CD4 T cells upon infection. However, in contrast to a well-established role for CD4 T cells in controlling persistent replication and a positive effect of IL-27 on their numbers, IL-27 promoted MCMV persistence in the salivary gland. This coincided with IL-27 mediated induction of IL-10 production in CD4 T cells. Moreover, IL-27 reduced expression of the transcription factor T-bet and restricted a cytotoxic phenotype in antiviral CD4 T cells. This is a highly intriguing result given the profound cytotoxic phenotype of CMV-specific CD4 T cells seen in humans and we established that dendritic cell derived IL-27 was responsible for this effect. Together, these data show that IL-27 regulates the number and effector functions of MCMV-specific CD4 T cells and could be targeted to enhance control of persistent/latent infection.

STAT2-Dependent Immune Responses Ensure Host Survival despite the Presence of a Potent Viral Antagonist.

ABSTRACTA pathogen encounter induces interferons, which signal via Janus kinases and STAT transcription factors to establish an antiviral state. However, the host and pathogens are situated in a continuous arms race which shapes host evolution toward optimized immune responses and the pathogens toward enhanced immune-evasive properties. Mouse cytomegalovirus (MCMV) counteracts interferon responses by pM27-mediated degradation of STAT2, which directly affects the signaling of type I as well as type III interferons. Using MCMV mutants lacking M27 and mice lacking STAT2, we studied the opposing relationship between antiviral activities and viral antagonism in a natural host-pathogen pair in vitro and in vivo. In contrast to wild-type (wt) MCMV, ΔM27 mutant MCMV was efficiently cleared from all organs within a few days in BALB/c, C57BL/6, and 129 mice, highlighting the general importance of STAT2 antagonism for MCMV replication. Despite this effective and relevant STAT2 antagonism, wt and STAT2-deficient mice exhibited fundamentally different susceptibilities to MCMV infections. MCMV replication was increased in all assessed organs (e.g., liver, spleen, lungs, and salivary glands) of STAT2-deficient mice, resulting in mortality during the first week after infection. Taken together, the results of our study reveal the importance of cytomegaloviral interferon antagonism for viral replication as well as a pivotal role of the remaining STAT2 activity for host survival. This mutual influence establishes a stable evolutionary standoff situation with fatal consequences when the equilibrium is disturbed.IMPORTANCE The host limits viral replication by the use of interferons (IFNs), which signal via STAT proteins. Several viruses evolved antagonists targeting STATs to antagonize IFNs (e.g., cytomegaloviruses, Zika virus, dengue virus, and several paramyxoviruses). We analyzed infections caused by MCMV expressing or lacking the STAT2 antagonist pM27 in STAT2-deficient and control mice to evaluate its importance for the host and the virus in vitro and in vivo. The inability to counteract STAT2 directly translates into exaggerated IFN susceptibility in vitro and pronounced attenuation in vivo. Thus, the antiviral activity mediated by IFNs via STAT2-dependent signaling drove the development of a potent MCMV-encoded STAT2 antagonist which became indispensable for efficient virus replication and spread to organs required for dissemination. Despite this clear impact of viral STAT2 antagonism, the host critically required the remaining STAT2 activity to prevent overt disease and mortality upon MCMV infection. Our findings highlight a remarkably delicate balance between host and virus.

FcγRI expression on macrophages is required for antibody-mediated tumor protection by cytomegalovirus-based vaccines.

Cytomegalovirus (CMV)-based vaccine vectors are promising vaccine platforms because they induce strong and long-lasting immune responses. Recently it has been shown that vaccination with a mouse CMV (MCMV) vector expressing the melanoma-specific antigen TRP2 (MCMV-TRP2) protects mice against outgrowth of TRP2-positive B16 melanoma tumors, and this protection was dependent on the induction of IgG antibodies. Here we demonstrate that, although mice lacking all receptors for the Fc part of IgG (FcγRs) develop normal IgG responses after MCMV-TRP2 vaccination, the protection against B16 melanoma was completely abrogated, indicating that FcγRs are indispensable in the downstream effector pathway of the polyclonal anti-TRP2 antibody response. By investigating compound FcγR-deficient mouse strains and by using immune cell type-specific cell ablation we show that the IgG antibody-mediated tumor protection elicited by MCMV-TRP2 mainly depends on FcγRI expression on macrophages, whereas FcγRIV plays only a modest role. Thus, tumor-specific antibody therapy might benefit from combination therapy that recruits FcγRI-expressing pro-inflammatory macrophages to the tumor micro-environment.

Anti-inflammatory consequences of bile acid accumulation in virus-infected bile duct ligated mice.

Cholestatic patients exhibiting high bile acid serum levels were reported to be more susceptible to bacterial and viral infections. Animal studies in bile duct ligated (BDL) mice suggest that cholestasis leads to an aggravation of hepatic bacterial infections. We have investigated the impact of cholestasis on mouse cytomegalovirus (MCMV)-induced immune responses and viral replication. While MCMV did not aggravate BDL-induced liver damage, BDL markedly reduced MCMV-triggered chemokine expression and immune cell recruitment to the liver. MCMV-infected BDL mice showed diminished trafficking of Ly6C+/F4/80+ myeloid cells and NK1.1+ NK cells to the liver compared to MCMV infected control mice. Moreover, virus-driven expression of CCL7, CCL12, CXCL9 and CXCL10 was clearly impaired in BDL- compared to sham-operated mice. Furthermore, production of the anti-inflammatory cytokine IL-10 was massively augmented in infected BDL mice. In contrast, intra- and extrahepatic virus replication was unaltered in BDL-MCMV mice when compared to sham-MCMV mice. Cholestasis in the BDL model severely impaired pathogen-induced chemokine expression in the liver affecting CCR2- and CXCR3-dependent cell trafficking. Cholestasis resulted in reduced recruitment of inflammatory monocytes and NK cells to the liver.

Transcription Factor IRF8 Orchestrates the Adaptive Natural Killer Cell Response

Natural killer (NK) cells are innate lymphocytes that display features of adaptive immunity during viral infection. Biallelic mutations in IRF8 have been reported to cause familial NK cell deficiency and susceptibility to severe viral infection in humans; however, the precise role of this transcription factor in regulating NK cell function remains unknown. Here, we show that cell-intrinsic IRF8 was required for NK-cell-mediated protection against mouse cytomegalovirus infection. During viral exposure, NK cells upregulated IRF8 through interleukin-12 (IL-12) signaling and the transcription factor STAT4, which promoted epigenetic remodeling of the Irf8 locus. Moreover, IRF8 facilitated the proliferative burst of virus-specific NK cells by promoting expression of cell-cycle genes and directly controlling Zbtb32, a master regulator of virus-driven NK cell proliferation. These findings identify the function and cell-type-specific regulation of IRF8 in NK-cell-mediated antiviral immunity and provide a mechanistic understanding of viral susceptibility in patients with IRF8 mutations.

Crk Adaptor Proteins Regulate NK Cell Expansion and Differentiation during Mouse Cytomegalovirus Infection

Natural killer cells are critical in the immune response to infection and malignancy. Prior studies have demonstrated that Crk family proteins can influence cell apoptosis, proliferation, and cell transformation. In this study, we investigated the role of Crk family proteins in mouse NK cell differentiation and host defense using a mouse CMV infection model. The number of NK cells, maturational state, and the majority of the NKR repertoire was similar in Crk x Crk-like (CrkL)-double-deficient and wild type NK cells. However, Crk family proteins were required for optimal activation, IFN-γ production, expansion, and differentiation of Ly49H+ NK cells, as well as host defense during mouse CMV infection. The diminished function of Crk x CrkL-double-deficient NK cells correlated with decreased phosphorylation of STAT4 and STAT1 in response to IL-12 and IFN-α stimulation, respectively. Together, our findings analyzing NK cell-specific Crk-deficient mice provide insights into the role of Crk family proteins in NK cell function and host defense.

Cytomegalovirus blockade of TRAIL-mediated type 1 ILC defenses regulates viral persistence

Abstract Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) regulates cellular apoptosis and inflammation, and growing evidence indicates a key role in antiviral defense. Cytomegalovirus (CMV, a b-herpesvirus) establishes a lifelong persistent/latent infection that depends upon viral subversion of host innate and adaptive defenses. We have shown that both human and mouse CMV inhibit signaling by the TRAIL death receptors (DR) through the use of specific viral proteins. MCMV m166 blocks TRAIL-DR expression and promotes early viral replication by evading TRAIL-mediated natural killer (NK) cell defenses. After one week of systemic replication, MCMV spreads to the salivary glands (SG), the major mucosal organ responsible for horizontal transmission of the virus. We show that a MCMVΔm166 mutant replicates at lower absolute levels in the SG, but, surprisingly, persists for much longer times. Recent evidence suggests a role of type 1 Innate Lymphoid cells (ILC1) in control/clearance of MCMV. We investigated the role of the 2 main ILC1 populations (conventional and tissue resident (tr) NK cells) in controlling MCMV SG persistence. Depleting ILC1 prior to infection, or infection of TRAIL−/− mice, restored MCMVΔm166 SG replication to normal levels. In addition, SG trNK cells display a unique phenotype and express high levels of TRAIL during MCMV infection when compared to their conventional counterparts. Together our data reveal a key role for m166 inhibition of TRAIL-DR expression in promoting viral persistence by blocking ILC1 effector functions mediated by TRAIL, highlighting this TNF-family cytokine as an important factor regulating innate defenses in mucosal tissues.

Brain-resident memory CD8+ Tcells induced by congenital CMV infection prevent brain pathology and virus reactivation.

Congenital HCMV infection is a leading infectious cause of long-term neurodevelopmental sequelae. Infection of newborn mice with mouse cytomegalovirus (MCMV) intraperitoneally is a well-established model of congenital human cytomegalovirus infection, which best recapitulates the hematogenous route of virus spread to brain and subsequent pathology. Here, we used this model to investigate the role, dynamics, and phenotype of CD8+ Tcells in the brain following infection of newborn mice. We show that CD8+ Tcells infiltrate the brain and form a pool of tissue-resident memory Tcells (TRM cells) that persist for lifetime. Adoptively transferred virus-specific CD8+ Tcells provide protection against primary MCMV infection in newborn mice, reduce brain pathology, and remain in the brain as TRM cells. Brain CD8+ TRM cells were long-lived, slowly proliferating cells able to respond to local challenge infection. Importantly, brain CD8+ TRM cells controlled latent MCMV and their depletion resulted in virus reactivation and enhanced inflammation in brain.

Cutting Edge: Divergent Requirement of T-Box Transcription Factors in Effector and Memory NK Cells.

The T-box transcription factors T-bet and Eomesodermin (Eomes) instruct discrete stages in NK cell development. However, their role in the immune response of mature NK cells against pathogens remains unexplored. We used an inducible deletion system to elucidate the cell-intrinsic role of T-bet and Eomes in mature NK cells during the course of mouse CMV infection. We show both T-bet and Eomes to be necessary for the expansion of virus-specific NK cells, with T-bet upregulation induced by IL-12 signaling and STAT4 binding to a conserved enhancer region upstream of the Tbx21 loci. Interestingly, our data suggest maintenance of virus-specific memory NK cell numbers and phenotype was dependent on T-bet, but not Eomes. These findings uncover a nonredundant and stage-specific influence of T-box transcription factors in the antiviral NK cell response.

The Contribution of Cytomegalovirus Infection to Immune Senescence Is Set by the Infectious Dose.

The relationship between human cytomegalovirus (HCMV) infections and accelerated immune senescence is controversial. Whereas some studies reported a CMV-associated impaired capacity to control heterologous infections at old age, other studies could not confirm this. We hypothesized that these discrepancies might relate to the variability in the infectious dose of CMV occurring in real life. Here, we investigated the influence of persistent CMV infection on immune perturbations and specifically addressed the role of the infectious dose on the contribution of CMV to accelerated immune senescence. We show in experimental mouse models that the degree of mouse CMV (MCMV)-specific memory CD8+ T cell accumulation and the phenotypic T cell profile are directly influenced by the infectious dose, and data on HCMV-specific T cells indicate a similar connection. Detailed cluster analysis of the memory CD8+ T cell development showed that high-dose infection causes a differentiation pathway that progresses faster throughout the life span of the host, suggesting a virus–host balance that is influenced by aging and infectious dose. Importantly, short-term MCMV infection in adult mice is not disadvantageous for heterologous superinfection with lymphocytic choriomeningitis virus (LCMV). However, following long-term CMV infection the strength of the CD8+ T cell immunity to LCMV superinfection was affected by the initial CMV infectious dose, wherein a high infectious dose was found to be a prerequisite for impaired heterologous immunity. Altogether our results underscore the importance of stratification based on the size and differentiation of the CMV-specific memory T cell pools for the impact on immune senescence, and indicate that reduction of the latent/lytic viral load can be beneficial to diminish CMV-associated immune senescence.

The Effects of IL10 and NK Cells on the Susceptibility to Mouse Cytomegalovirus in BALB/c Mice despite the Compensation of IFNγ

Objective: The aim of this study was to determine which factors lead to the susceptibility to mouse cytomegalovirus (MCMV) in the spleens of BALB/c mice. Methods: BALB/c and C57BL/6 mice were randomly divided into a control group and an infection group and sacrificed on day 0, 1, 3, 7, 14, and 28 postinfection. The cytotoxicity of NK cells was determined by evaluating lactate dehydrogenase contents. Flow cytometry was used to analyze activated NK cells, IFNγ⁺ NK cells, and total NK cells in the spleen. The pathological changes of spleens in each group were analyzed by HE staining. The expression of IL10, IL18, IFNγ, Thpok, and IFNβ of spleens was determined by quantitative reverse transcriptase PCR. The viral loads of MCMV in spleens and salivary glands were also detected. Results: We found that spleen NK cells and IL10 in C57BL/6 mice possessed more powerful immunity to MCMV than BALB/c mice. In BALB/c mice, combined effects of the cytotoxicity of NK cells and IFNγ in spleens still ended up with deficient control of infection. Conclusion: The functional shortage of NK cells and inappropriate expression of IL10 result in the susceptibility to MCMV in BALB/c mice.

Viral Replication Assay in Bone Marrow-Derived Macrophages.

The selection of macrophages as a cell type for investigating virus-host interactions is based on cellular tropism of the virus during infection as well as contribution of these cells to pathogenesis in the host. In response to mouse cytomegalovirus (MCMV) infection, bone marrow-resident monocytes that mobilize to infected tissues to differentiate into macrophages and dendritic cells are hijacked in order to facilitate viral persistence. These cells contribute significantly to MCMV biology and, thus, are actively recruited by the virus-encoded chemokine. In this chapter, we provide detailed methodologies employed in our laboratory to assess MCMV replication in bone marrow-derived macrophages.

Comparison of human coagulation factor VIII expression directed by cytomegalovirus and mammary gland-specific promoters in HC11 cells and transgenic mice: Retraction.

Comparison of human coagulation factor VIII expression directed by cytomegalovirus and mammary gland-specific promoters in HC11 cells and transgenic mice: Retraction.

Continuous activity of Foxo1 is required to prevent anergy and maintain the memory state of CD8+ T cells.

Upon infection with an intracellular pathogen, cytotoxic CD8+ T cells develop diverse differentiation states characterized by function, localization, longevity, and the capacity for self-renewal. The program of differentiation is determined, in part, by FOXO1, a transcription factor known to integrate extrinsic input in order to specify survival, DNA repair, self-renewal, and proliferation. At issue is whether the state of T cell differentiation is specified by initial conditions of activation or is actively maintained. To study the spectrum of T cell differentiation, we have analyzed an infection with mouse cytomegalovirus, a persistent-latent virus that elicits different cytotoxic T cell responses characterized as acute resolving or inflationary. Our results show that FOXO1 is continuously required for all the phenotypic characteristics of memory-effector T cells such that with acute inactivation of the gene encoding FOXO1, T cells revert to a short-lived effector phenotype, exhibit reduced viability, and manifest characteristics of anergy.

The C-terminal part of the human cytomegalovirus terminase subunit pUL51 is central for terminase complex assembly.

The cleavage and packaging of the human cytomegalovirus (HCMV) genome is accomplished by the viral terminase, comprising pUL56 and pUL89, and the recently identified pUL51 subunit. Since knowledge about pUL51 is scarce, we aimed at identifying pUL51 domains that are important for terminase assembly. In silico analysis suggested that the N-terminal half of pUL51 is intrinsically disordered, and that α-helices are present in the C-terminal part. Linker-scanning mutagenesis of pUL51 in the context of the viral genome revealed that amino acid insertions into the predicted α-helices are not compatible with viral growth, whereas upon mutagenesis of the putatively disordered parts interaction with pUL56 and pUL89 was retained and viral progeny was produced. Replacement of pUL51 with the closely related M51 protein of mouse cytomegalovirus did not lead to viable virus, indicating that M51 cannot substitute for pUL51, and swapping the M51 and UL51 N- and C-termini demonstrated the critical role of the pUL51 C-terminal part in building the terminase complex. Notably, the pUL51 C-terminus alone turned out to be sufficient to enable terminase assembly, its nuclear localization and plaque formation. Using HCMV mutants expressing differently tagged pUL51 versions, we did not detect oligomerization of pUL51, as has been proposed for the pUL51 orthologues of other herpesviruses. These data provide an insight into the interaction of pUL51 with the other two terminase components, and provide the basis for unravelling the mode of action of novel antiviral drugs targeting the HCMV terminase.

The M25 gene products are critical for the cytopathic effect of mouse cytomegalovirus

Cell rounding is a hallmark of the cytopathic effect induced by cytomegaloviruses. By screening a panel of deletion mutants of mouse cytomegalovirus (MCMV) a mutant was identified that did not elicit cell rounding and lacked the ability to form typical plaques. Altered cell morphology was assigned to the viral M25 gene. We detected an early 2.8 kb M25 mRNA directing the synthesis of a 105 kDa M25 protein, and confirmed that a late 3.1 kb mRNA encodes a 130 kDa M25 tegument protein. Virions lacking the M25 tegument protein were of smaller size because the tegument layer between capsid and viral envelope was reduced. The ΔM25 mutant did not provoke the rearrangement of the actin cytoskeleton observed after wild-type MCMV infection, and isolated expression of the M25 proteins led to cell size reduction, confirming that they contribute to the morphological changes. Yields of progeny virus and cell-to-cell spread of the ΔM25 mutant in vitro were diminished and replication in vivo was impaired. The identification of an MCMV gene involved in cell rounding provides the basis for investigating the role of this cytopathic effect in CMV pathogenesis.

ILC1 Confer Early Host Protection at Initial Sites of Viral Infection

Infection is restrained by the concerted activation oftissue-resident and circulating immune cells. Whether tissue-resident lymphocytes confer early antiviral immunity at local sites of primary infection prior to the initiation of circulating responses is not well understood. Furthermore, the kinetics of initial antiviral responses at sites of infection remain unclear. Here, we show that tissue-resident type 1 innate lymphoid cells (ILC1) serve an essential early role in host immunity through rapid production of interferon (IFN)-γ following viral infection. Ablation of Zfp683-dependent liver ILC1 lead to increased viral load in the presence of intact adaptive and innate immune cells critical for mouse cytomegalovirus (MCMV) clearance. Swift production of interleukin (IL)-12 by tissue-resident XCR1+ conventional dendritic cells (cDC1) promoted ILC1 production of IFN-γ in a STAT4-dependent manner to limit early viral burden. Thus, ILC1 contribute an essential role in viral immunosurveillance at sites of initial infection in response to local cDC1-derived proinflammatory cytokines.