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Cytomegaloviruses (CMVs), members of the β-subfamily of the herpesvirus family, have co-speciated with their respective mammalian hosts resulting in a mutual virus-host adaptation reflected by sets of 'private' viral genes that a particular CMV species does not share with other CMVs and that define the host-species specificity of CMVs. Nonetheless, based on "biological convergence" in evolution, fundamental rules in viral pathogenesis and immune control are functionally analogous between different virus-host pairs. Therefore, the mouse model of infection with murine CMV (mCMV) has revealed generally valid principles of CMV-host interactions. Specifically, the mouse model has paved the way to cellular immunotherapy of CMV disease in immunocompromised recipients of hematopoietic cell transplantation (HCT). Precisely in the context of HCT, however, current view assumes that there exists a major difference between hCMV and mCMV regarding "latent virus reservoirs" in that only hCMV establishes latency in hematopoietic lineage cells (HLCs), whereas mCMV establishes latency in endothelial cells. This would imply that only hCMV can reactivate from transplanted HLCs of a latently infected donor. In addition, as viral transcriptional activity during latency is discussed as a driver of clonal T-cell expansion over lifetime, a phenomenon known as "memory inflation", it is important to know if hCMV and mCMV establish latency in the same cell type(s) for imprinting the immune system. Here, we review the currently available evidence to propose that the alleged difference in latent virus reservoirs between hCMV and mCMV may rather relate to a difference in the focus of research. While studies on hCMV latency in HLCs likely described a non-canonical, transient type-2 latency, studies in the mouse model focussed on canonical, lifelong type-1 latency.

As an immune evasion mechanism, cytomegaloviruses (CMVs) have evolved proteins that interfere with cell surface trafficking of MHC class-I (MHC-I) molecules to tone down recognition by antiviral CD8 T cells. This interference can affect the trafficking of recently peptide-loaded MHC-I from the endoplasmic reticulum to the cell surface, thus modulating the presentation of viral peptides, as well as the recycling of pre-existing cell surface MHC-I, resulting in reduction of the level of overall MHC-I cell surface expression. Murine cytomegalovirus (mCMV) was paradigmatic in that it led to the discovery of this immune evasion strategy of CMVs. Members of its m02-m16 gene family code for type-I transmembrane glycoproteins, proven or predicted, most of which carry cargo sorting motifs in their cytoplasmic, C-terminal tail. For the m06 gene product m06 (gp48), the cargo has been identified as being MHC-I, which is linked by m06 to cellular adapter proteins AP-1A and AP-3A through the dileucine motif EPLARLL. Both APs are involved in trans-Golgi network (TGN) cargo sorting and, based on transfection studies, their engagement by the dileucine motif was proposed to be absolutely required to prevent MHC-I exposure at the cell surface. Here, we have tested this prediction in an infection system with the herein newly described recombinant virus mCMV-m06AA, in which the dileucine motif is destroyed by replacing EPLARLL with EPLARAA. This mutation has a phenotype in that the transition of m06-MHC-I complexes from early endosomes (EE) to late endosomes (LE)/lysosomes for degradation is blocked. Consistent with the binding of the MHC-I α-chain to the luminal domain of m06, the m06-mediated disposal of MHC-I did not require the β2m chain of mature MHC-I. Unexpectedly, however, disconnecting MHC-I cargo from AP-1A/3A by the motif mutation in m06 had no notable rescuing impact on overall cell surface MHC-I, though it resulted in some improvement of the presentation of viral antigenic peptides by recently peptide-loaded MHC-I. Thus, the current view on the mechanism by which m06 mediates immune evasion needs to be revised. While the cargo sorting motif is critically involved in the disposal of m06-bound MHC-I in the endosomal/lysosomal pathway at the stage of EE to LE transition, this motif-mediated disposal is not the critical step by which m06 causes immune evasion. We rather propose that engagement of AP-1A/3A by the cargo sorting motif in m06 routes the m06-MHC-I complexes into the endosomal pathway and thereby detracts them from the constitutive cell surface transport.

Roizman's definition of herpesviral latency, which applies also to cytomegaloviruses (CMVs), demands maintenance of reactivation-competent viral genomes after clearance of productive infection. It is more recent understanding that failure to complete the productive viral cycle for virus assembly and release does not imply viral gene silencing at all genetic loci and all the time. It rather appears that CMV latency is transcriptionally "noisy" in that silenced viral genes get desilenced from time to time in a stochastic manner, leading to "transcripts expressed in latency" (TELs). If a TEL happens to code for a protein that contains a CD8 T cell epitope, protein processing can lead to the presentation of the antigenic peptide and restimulation of cognate CD8 T cells during latency. This mechanism is discussed as a potential driver of epitope-selective accumulation of CD8 T cells over time, a phenomenon linked to CMV latency and known as "memory inflation" (MI). So far, expression of an epitope-encoding TEL was shown only for the major immediate-early (MIE) gene m123/ie1 of murine cytomegalovirus (mCMV), which codes for the prototypic MI-driving antigenic peptide YPHFMPTNL that is presented by the MHC class-I molecule Ld. The only known second MI-driving antigenic peptide of mCMV in the murine MHC haplotype H-2d is AGPPRYSRI presented by the MHC-I molecule Dd. This peptide is very special in that it is encoded by the early (E) phase gene m164 and by an overlapping immediate-early (IE) transcript governed by a promoter upstream of m164. If MI is driven by presentation of TEL-derived antigenic peptides, as the hypothesis says, one should find corresponding TELs. We show here that E-phase and IE-phase transcripts that code for the MI-driving antigenic peptide AGPPRYSRI are independently and stochastically expressed in latently infected lungs.

Reactivation of latent cytomegalovirus (CMV) endangers the therapeutic success of hematopoietic cell transplantation (HCT) in tumor patients due to cytopathogenic virus spread that leads to organ manifestations of CMV disease, to interstitial pneumonia in particular. In cases of virus variants that are refractory to standard antiviral pharmacotherapy, immunotherapy by adoptive cell transfer (ACT) of virus-specific CD8+ T cells is the last resort to bridge the “protection gap” between hematoablative conditioning for HCT and endogenous reconstitution of antiviral immunity. We have used the well-established mouse model of CD8+ T-cell immunotherapy by ACT in a setting of experimental HCT and murine CMV (mCMV) infection to pursue the concept of improving the efficacy of ACT by therapeutic vaccination (TherVac) post-HCT. TherVac aims at restimulation and expansion of limited numbers of transferred antiviral CD8+ T cells within the recipient. Syngeneic HCT was performed with C57BL/6 mice as donors and recipients. Recipients were infected with recombinant mCMV (mCMV-SIINFEKL) that expresses antigenic peptide SIINFEKL presented to CD8+ T cells by the MHC class-I molecule Kb. ACT was performed with transgenic OT-I CD8+ T cells expressing a T-cell receptor specific for SIINFEKL-Kb. Recombinant human CMV dense bodies (DB-SIINFEKL), engineered to contain SIINFEKL within tegument protein pUL83/pp65, served for vaccination. DBs were chosen as they represent non-infectious, enveloped, and thus fusion-competent subviral particles capable of activating dendritic cells and delivering antigens directly into the cytosol for processing and presentation in the MHC class-I pathway. One set of our experiments documents the power of vaccination with DBs in protecting the immunocompetent host against a challenge infection. A further set of experiments revealed a significant improvement of antiviral control in HCT recipients by combining ACT with TherVac. In both settings, the benefit from vaccination with DBs proved to be strictly epitope-specific. The capacity to protect was lost when DBs included the peptide sequence SIINFEKA lacking immunogenicity and antigenicity due to C-terminal residue point mutation L8A, which prevents efficient proteasomal peptide processing and binding to Kb. Our preclinical research data thus provide an argument for using pre-emptive TherVac to enhance antiviral protection by ACT in HCT recipients with diagnosed CMV reactivation.

While establishing a regional SARS-Cov-2 variant surveillance by genome sequencing, we have identified three infected individuals in a clinical setting (two long-term hospitalized patients and a nurse) that shared the spike N501Y mutation within a genotype background distinct from the current viral variants of concern. We suggest that the adaptive N501Y mutation, known to increase SARS-CoV-2 transmissibility, arose by convergent evolution around December in Mainz, Germany. Hospitalized patients with a compromised immune system may be a potential source of novel viral variants, which calls for monitoring viral evolution by genome sequencing in clinical settings.

Several rapid antigen tests (RATs) for the detection of SARS-CoV-2 were evaluated recently. However, reliable performance data for laboratory-based, high-throughput antigen tests are lacking. Therefore and in response to a short-term shortage of PCR reagents, we evaluated DiaSorin's LIAISON SARS-CoV-2 antigen test in comparison to RT-qPCR, and concerning the application of screening non-COVID-19 patients on hospital admission. Applying the manufacturer-recommended cut-off of 200 arbitrary units (AU/mL) the specificity of the LIAISON Test was 100%, the overall analytical sensitivity 40.2%. Lowering the cut-off to 100 AU/mL increased the sensitivity to 49.7% and decreased the specificity to 98.3%. Confining the analysis to samples with an RT-qPCR result

Murine models of cytomegalovirus (CMV) infection have revealed an exceptional kinetics of the immune response. After resolution of productive infection, transient contraction of the viral epitope-specific CD8 T-cell pool was found to be followed by a pool expansion specific for certain viral epitopes during non-productive &lsquo, latent&rsquo, infection. This phenomenon, known as &lsquo, memory inflation&rsquo, (MI), was found to be based on inflationary KLRG1+CD62L&minus, effector-memory T cells (iTEM) that depend on repetitive restimulation. MI gained substantial interest for employing CMV as vaccine vector by replacing MI-driving CMV epitopes with foreign epitopes for generating high numbers of protective memory cells specific for unrelated pathogens. The concept of an MI-driving CMV vector is questioned by human studies disputing MI in humans. A bias towards MI in experimental models may have resulted from systemic infection. We have here studied local murine CMV infection as a route that is more closely matching routine human vaccine application. Notably, KLRG1&minus, CD62L+ central memory T cells (TCM) and conventional KLRG1&minus, CD62L&minus, effector memory T cells (cTEM) were found to expand, associated with &lsquo, avidity maturation&rsquo, whereas the pool size of iTEM steadily declined over time. The establishment of high avidity CD8 T-cell central memory encourages one to pursue the concept of CMV vector-based vaccines.

Hematoablative treatment followed by hematopoietic cell transplantation (HCT) for reconstituting the co-ablated immune system is a therapeutic option to cure aggressive forms of hematopoietic malignancies. In cases of family donors or unrelated donors, immunogenetic mismatches in major histocompatibility complex (MHC) and/or minor histocompatibility (minor-H) loci are unavoidable and bear a risk of graft-vs.-host reaction and disease (GvHR/D). Transient immunodeficiency inherent to the HCT protocol favors a productive reactivation of latent cytomegalovirus (CMV) that can result in multiple-organ CMV disease. In addition, there exists evidence from a mouse model of MHC class-I-mismatched GvH-HCT to propose that mismatches interfere with an efficient reconstitution of antiviral immunity. Here we used a mouse model of MHC-matched HCT with C57BL/6 donors and MHC-congenic BALB.B recipients that only differ in polymorphic autosomal background genes, including minor-H loci coding for minor-H antigens (minor-HAg). Minor-HAg mismatch is found to promote lethal CMV disease in absence of a detectable GvH response to an immunodominant minor-HAg, the H60 locus-encoded antigenic peptide LYL8. Lethality of infection correlates with inefficient reconstitution of viral epitope-specific CD8+ T cells. Notably, lethality is prevented and control of cytopathogenic infection is restored when viral antigen presentation is enhanced by deletion of immune evasion genes from the infecting virus. We hypothesize that any kind of mismatch in GvH-HCT can induce “non-cognate transplantation tolerance” that dampens not only a mismatch-specific GvH response, which is beneficial, but adversely affects also responses to mismatch-unrelated antigens, such as CMV antigens in the specific case, with the consequence of lethal CMV disease.

The complexity of host-associated microbial ecosystems requires host-specific reference catalogs to survey the functions and diversity of these communities. We generate a comprehensive resource, the integrated mouse gut metagenome catalog (iMGMC), comprising 4.6 million unique genes and 660 metagenome-assembled genomes (MAGs), many (485 MAGs, 73%) of which are linked to reconstructed full-length 16S rRNA gene sequences. iMGMC enables unprecedented coverage and taxonomic resolution of the mouse gut microbiota; i.e., more than 92% of MAGs lack species-level representatives in public repositories (

The memory CD8 T-cell pool must select for clones that bind immunodominant epitopes with high affinity to efficiently counter reinfection. At the same time, it must retain a level of clonal diversity to allow recognition of pathogens with mutated epitopes. How the level of diversity within the memory pool is controlled is unclear, especially in the context of a selective drive for antigen affinity. We find that preservation of clones that bind the activating antigen with low affinity depends on expression of the transcription factor Eomes in the first days after antigen encounter. Eomes is induced at low activating signal strength and directly drives transcription of the prosurvival protein Bcl-2. At higher signal intensity, T-bet is induced which suppresses Bcl-2 and causes a relative survival advantage for cells of low affinity. Clones activated with high-affinity antigen form memory largely independent of Eomes and have a proliferative advantage over clones that bind the same antigen with low affinity. This causes high-affinity clones to prevail in the memory pool, despite their relative survival deficit. Genetic or therapeutic targeting of the Eomes/Bcl-2 axis reduces the clonal diversity of the memory pool, which diminishes its ability to respond to pathogens carrying mutations in immunodominant epitopes. Thus, we demonstrate on a molecular level how sufficient diversity of the memory pool is established in an environment of affinity-based selection., This study shows that the diversity of the memory CD8 T-cell pool is regulated by the transcription factor Eomes, which drives transcription of the pro-survival protein Bcl-2. Genetic or therapeutic targeting of the Eomes/Bcl-2 axis reduces the clonal diversity of the memory pool.

Despite a broad cell-type tropism, cytomegalovirus (CMV) is an evidentially pulmonary pathogen. Predilection for the lungs is of medical relevance in immunocompromised recipients of hematopoietic cell transplantation, in whom interstitial CMV pneumonia is a frequent and, if left untreated, fatal clinical manifestation of human CMV infection. A conceivable contribution of CMV to airway diseases of other etiology is an issue that so far attracted little medical attention. As the route of primary CMV infection upon host-to-host transmission in early childhood involves airway mucosa, coincidence of CMV airway infection and exposure to airborne environmental antigens is almost unavoidable. For investigating possible consequences of such a coincidence, we established a mouse model of airway co-exposure to CMV and ovalbumin (OVA) representing a protein antigen of an inherently low allergenic potential. Accordingly, intratracheal OVA exposure alone failed to sensitize for allergic airway disease (AAD) upon OVA aerosol challenge. In contrast, airway infection at the time of OVA sensitization predisposed for AAD that was characterized by airway inflammation, IgE secretion, thickening of airway epithelia, and goblet cell hyperplasia. This AAD histopathology was associated with a T helper type 2 (Th2) transcription profile in the lungs, including IL-4, IL-5, IL-9, and IL-25, known inducers of Th2-driven AAD. These symptoms were all prevented by a pre-challenge depletion of CD4+ T cells, but not of CD8+ T cells. As to the underlying mechanism, murine CMV activated migratory CD11b+ as well as CD103+ conventional dendritic cells (cDCs), which have been associated with Th2 cytokine-driven AAD and with antigen cross-presentation, respectively. This resulted in an enhanced OVA uptake and recruitment of the OVA-laden cDCs selectively to the draining tracheal lymph nodes for antigen presentation. We thus propose that CMV, through activation of migratory cDCs in the airway mucosa, can enhance the allergenic potential of otherwise poorly allergenic environmental protein antigens., Author summary From an epidemiological perspective, natural host-to-host transmission of human CMV mostly occurs in early childhood through saliva from virus-shedding intimate contact persons, such as family members or peers. The oronasal route of transmission involves also the airway mucosa and airway-draining lymph nodes. Almost unavoidably, CMV airway infection coincides with airway exposure to environmental antigens, which include potent classical allergens but also protein antigens that have low-to-no allergenic potential on their own. Ovalbumin (OVA), when administered as a purified protein, can serve as a well-studied model antigen for only poorly allergenic environmental antigens. In a murine model of airway exposure to OVA for allergenic sensitization, we have addressed the question if a simultaneous airway infection with murine CMV (mCMV) can promote allergic airway disease (AAD) elicited by challenge exposure to OVA. As anticipated by the model design, exposure to OVA alone did not sensitize for an allergic response to challenge exposure, nor did mCMV infection alone. Notably, based on viral activation of antigen uptake by DCs, both combined sensitized for a type 2 CD4+ T helper (Th2) cell-driven AAD histopathology. This is a novel aspect in CMV pathobiology with the medical relevance that CMV airway infection enlarges the spectrum of environmental allergens.

NKG2D is an activating receptor that is expressed on most cytotoxic cells of the immune system, including NK cells, γδ and CD8+ T cells. It is still a matter of debate whether and how NKG2D mediates priming of CD8+ T cells in vivo, due to a lack of studies where NKG2D is eliminated exclusively in these cells. Here we studied the impact of NKG2D on effector CD8+ T-cell formation. NKG2D-deficiency that is restricted to murine CD8+ T cells did not impair antigen-specific T-cell expansion following mCMV and LCMV infection, but reduced their capacity to produce cytokines. Upon infection, conventional dendritic cells induce NKG2D ligands, which drive cytokine production on CD8+ T cells via the Dap10 signaling pathway. T-cell development, homing and proliferation were not affected by NKG2D deficiency and cytotoxicity was only impaired when strong T-cell receptor stimuli were used. Transfer of antigen-specific CD8+ T cells demonstrated that NKG2D-deficiency attenuated their capacity to reduce viral loads. The inability of NKG2D-deficient cells to produce cytokines could be overcome with injection of IL-15 super-agonist during priming. In summary, our data shows that NKG2D has a non-redundant role in priming of CD8+ T cells to produce antiviral cytokines. Upon viral infection, classical Dendritic cells induce expression of the NKG2D ligand H60. NKG2D stimulation during priming enhances the ability of CD8 T cells to produce cytokines but not increases cytotoxic potential upon T cell receptor engagement in the periphery.

Background: Coronavirus disease-2019 (COVID-19) triggers systemic infection with involvement of the respiratory tract. There are some patients developing haemostatic abnormalities during their infection with a considerably increased risk of death. Materials and Methods: Patients (n = 85) with SARS-CoV-2 infection attending the University Medical Center, Mainz, from 3 March to 15 May 2020 were retrospectively included in this study. Data regarding demography, clinical features, treatment and laboratory parameters were analyzed. Twenty patients were excluded for assessment of disseminated intravascular coagulation (DIC) and thrombotic microangiopathy (TMA) due to lack of laboratory data. Results: COVID-19 patients (n = 65) were investigated, 19 with uncomplicated, 29 with complicated, and 17 with critical course, nine (13.8%) died. Seven patients showed overt DIC according to the ISTH criteria. The fibrinogen levels dropped significantly in these patients, although not below 100 mg/dl. Hallmarks of TMA, such as thrombocytopenia and microangiopathic haemolytic anaemia, were not detected in any of our COVID-19 patients. ADAMTS13 activity was mildly to moderately reduced in 4/22 patients, all having strongly elevated procalcitonin levels. Conclusion: DIC occurred in 7/65 COVID-19 patients but fibrinogen and platelet consumption were compensated in almost all. ADAMTS13 assays excluded TTP and hallmarks of classic TMA were absent in all investigated patients. We hypothesize that the lacking erythrocyte fragmentation and only mild platelet consumption in severe COVID-19 are due to a microangiopathy predominantly localized to the alveolar microcirculation with a low blood pressure gradient.

Cytomegaloviruses (CMVs) are master manipulators of the host immune response. Here, we reveal that the murine CMV (MCMV) protein m152 specifically targets the type I interferon (IFN) response by binding to stimulator of interferon genes (STING), thereby delaying its trafficking to the Golgi compartment from where STING initiates type I IFN signaling. Infection with an MCMV lacking m152 induced elevated type I IFN responses and this leads to reduced viral transcript levels both in vitro and in vivo This effect is ameliorated in the absence of STING Interestingly, while m152 inhibits STING-mediated IRF signaling, it did not affect STING-mediated NF-κB signaling. Analysis of how m152 targets STING translocation reveals that STING activates NF-κB signaling already from the ER prior to its trafficking to the Golgi. Strikingly, this response is important to promote early MCMV replication. Our results show that MCMV has evolved a mechanism to specifically antagonize the STING-mediated antiviral IFN response, while preserving its pro-viral NF-κB response, providing an advantage in the establishment of an infection.

Initial virus entry into cells of host organs and subsequent spread of viral progeny between tissue cells are events fundamental to viral pathogenesis. Glycoprotein complexes inserted in the virion envelope are critically involved in the cell entry process. Here we review and discuss recent work that has shed light on the in vivo role of the trimeric glycoprotein complex gH/gL/gO of murine cytomegalovirus (mCMV) as a model to propose the role of the corresponding complex of human CMV, for which experimental studies in vivo are not feasible due to the host species specificity of CMVs and evident ethical constraints. A novel approach combining gO transcomplementation of a genetically gO-deficient virus and a mathematical log-linear regression analysis of the viral multiplication kinetics in host tissues revealed a critical role of mCMV gH/gL/gO only in first target cell entry of virions arriving with the circulation, whereas intra-tissue spread proceeded unaffected also in the absence of gH/gL/gO. These findings predict that targeting gO for an antiviral intervention may be of prophylactic value in preventing the seeding of virus to organs, but will likely fail to interfere with an established primary organ infection or with recurrent infection after virus reactivation from latency within tissue cells. The demonstration in the murine model of alternative gH/gL complexes gH/gL/gO and gH/gL/MCK-2, substituting one another in a redundant fashion for securing viral spread in tissues, has the medically interesting bearing that targeting the gH/gL core complex directly may be a promising approach to preventing primary, established, and recurrent CMV infections.

Human cytomegalovirus (CMV) is one of the largest viruses known to cause human diseases. Chronic CMV infection, as defined by anti-CMV IgG serology, increases with age and is highly prevalent in older adults. It has complex biology with significant immunologic and health consequences. This article aims to summarize research findings presented at the 6th International Workshop on CMV and Immunosenescence that relate to advances in the areas of CMV tropism, latency, CMV manipulation of cell metabolism, and T cell memory inflation, as well as novel diagnostic evaluation and translational research of chronic CMV infection in older adults. Information summarized here represents the current state of knowledge in these important fields. Investigators have also identified a number of areas that deserve further and more in-depth investigation, including building more precise parallels between mouse CMV (mCMV) and human CMV (HCMV) research. It is hoped that this article will also stimulate engaging discussion on strategies and direction to advance the science to the next level.

Interstitial pneumonia is a life-threatening clinical manifestation of human cytomegalovirus (hCMV) infection. In particular, it can be deadly in patients with hematopoietic malignancies who undergo hematopoietic cell transplantation (HCT) in whom a ‘window of risk’, which is defined by transient immunodeficiency, occurs between hematoablative therapeutic treatment and immunological reconstitution. As few clinical studies have addressed the underlying mechanisms for this phenomenon, a mouse model of HCT and murine cytomegalovirus (mCMV) infection has been established and has revealed a key role for antiviral CD8+ T cells in controlling pulmonary infections. Using this mouse model, recent studies have provided evidence for the role of mast cells (MC) in more efficiently recruiting antiviral CD8+ T cells to CMV-infected lungs in a process that involves the release of CCL5, a chemokine. MC were found to be direct cellular targets of murine CMV infection and could be activated and degranulated by two mechanisms: the rapid degranulation requiring TLR3/TRIF signaling in non-MC cells and a delayed TLR3/TRIF-independent mechanism. Here, we provide evidence showing that TLR3/TRIF-independent MC activation by CMV is the dominant mechanism in controlling pulmonary infection.

Novel approaches of localization microscopy have opened new insights into the molecular nano-cosmos of cells. We applied a special embodiment called spectral position determination microscopy (SPDM) that has the advantage to run with standard fluorescent dyes or proteins under standard preparation conditions. Pointillist images with a resolution in the order of 10 nm can be obtained by SPDM. Therefore, vector pEYFP-m164, encoding the murine cytomegalovirus glycoprotein gp36.5/m164 fused to enhanced yellow fluorescent protein, was transiently transfected into COS-7 cells. This protein shows exceptional intracellular trafficking dynamics, moving within the endoplasmic reticulum (ER) and outer nuclear membrane. The molecular positions of gp36.5/m164 were visualized and determined by SPDM imaging. From the position point patterns of the protein molecules, their arrangements were quantified by next neighbour distance analyses. Three different structural arrangements were discriminated: (a) a linear distribution along the membrane, (b) a highly structured distribution in the ER, and (c) a homogenous distribution in the cellular cytoplasm. The results indicate that the analysis of next neighbour distances on the nano-scale allows the identification and discrimination of different structural arrangements of molecules within their natural cellular environment.

The type I interferon (IFN) response is imperative for the establishment of the early antiviral immune response. Here we report the identification of the first type I IFN antagonist encoded by murine cytomegalovirus (MCMV) that shuts down signaling following pattern recognition receptor (PRR) sensing. Screening of an MCMV open reading frame (ORF) library identified M35 as a novel and strong negative modulator of IFNβ promoter induction following activation of both RNA and DNA cytoplasmic PRR. Additionally, M35 inhibits the proinflammatory cytokine response downstream of Toll-like receptors (TLR). Using a series of luciferase-based reporters with specific transcription factor binding sites, we determined that M35 targets NF-κB-, but not IRF-mediated, transcription. Expression of M35 upon retroviral transduction of immortalized bone marrow-derived macrophages (iBMDM) led to reduced IFNβ transcription and secretion upon activation of stimulator of IFN genes (STING)-dependent signaling. On the other hand, M35 does not antagonize interferon-stimulated gene (ISG) 56 promoter induction or ISG transcription upon exogenous stimulation of the type I IFN receptor (IFNAR). M35 is present in the viral particle and, upon MCMV infection of fibroblasts, is immediately shuttled to the nucleus where it exerts its immunomodulatory effects. Deletion of M35 from the MCMV genome and hence from the viral particle resulted in elevated type I IFN transcription and secretion in vitro and in vivo. In the absence of M35, lower viral titers are observed during acute infection of the host, and productive infection in the salivary glands was not detected. In conclusion, the M35 protein is released by MCMV immediately upon infection in order to deftly inhibit the antiviral type I IFN response by targeting NF-κB-mediated transcription. The identification of this novel viral protein reinforces the importance of timely countermeasures in the complex relationship between virus and host., Author summary The herpesvirus cytomegalovirus can cause severe morbidity in immunosuppressed people and poses a much greater global problem in the context of congenital infections than the Zika virus. To establish infection, cytomegalovirus needs to modulate the antiviral immune response of its host. One of the first lines of defense against viral infections is the type I interferon response which is activated by cellular sensors called pattern recognition receptors. These receptors sense viral entry and rapidly induce the transcription of type I interferons, which are instrumental for the induction of an antiviral state in infected and surrounding cells. We have identified the first viral protein encoded by murine cytomegalovirus, the M35 protein, that counteracts type I interferon transcription downstream of multiple pattern recognition receptors. We found that this viral countermeasure occurs shortly after viral entry into the host cell, as M35 is delivered with the viral particle. M35 then localizes to the nucleus where it modulates NF-κB-mediated transcription. In vivo, murine cytomegalovirus deficient of the M35 protein replicates to lower levels in spleen and liver and cannot establish a productive infection in the salivary glands, which is a key site of viral transmission, highlighting the important role of M35 for the establishment of infection. Our study provides novel insights into the complex interaction between cytomegalovirus and the innate immune response of its host.

Designing of CD8 T cell vaccines which would provide protection against tumors is still considered a great challenge in immunotherapy. Here we show a robust potential of a cytomegalovirus (CMV) vector expressing the NKG2D ligand RAE-1γ as CD8 T cell-based vaccine against malignant tumors. Immunization with the CMV vector expressing RAE-1γ delayed tumor growth or even provided complete protection against tumor challenge in both prophylactic and therapeutic settings. Moreover, a potent tumor control in mice vaccinated with this vector can be further enhanced by blocking the immune checkpoints TIGIT and PD-1. Expression of RAE-1γ by the CMV vector potentiated expansion of KLRG1+ CD8 T cells with enhanced effector properties. This vaccination was even more efficient in neonatal mice, resulting in the expansion and long-term maintenance of epitope-specific CD8 T cells conferring robust resistance against tumor challenge. Our data show that immunomodulation of CD8 T cell responses promoted by herpesvirus expressing a ligand for NKG2D receptor can provide a powerful platform for the prevention and treatment of CD8 T cell-sensitive tumors.

The receptor-like protein tyrosine phosphatase CD45 is expressed on the surface of cells of hematopoietic origin and has a pivotal role for the function of these cells in the immune response. Here we report that following infection of macrophages with mouse cytomegalovirus (MCMV) the cell surface expression of CD45 is drastically diminished. Screening of a set of MCMV deletion mutants allowed us to identify the viral gene m42 of being responsible for CD45 down-modulation. Moreover, expression of m42 independent of viral infection upon retroviral transduction of the RAW264.7 macrophage cell line led to comparable regulation of CD45 expression. In immunocompetent mice infected with an m42 deletion mutant lower viral titers were observed in all tissues examined when compared to wildtype MCMV, indicating an important role of m42 for viral replication in vivo. The m42 gene product was identified as an 18 kDa protein expressed with early kinetics and is predicted to be a tail-anchored membrane protein. Tracking of surface-resident CD45 molecules revealed that m42 induces internalization and degradation of CD45. The observation that the amounts of the E3 ubiquitin ligases Itch and Nedd4 were diminished in cells expressing m42 and that disruption of a PY motif in the N-terminal part of m42 resulted in loss of function, suggest that m42 acts as an activator or adaptor for these Nedd4-like ubiquitin ligases, which mark CD45 for lysosomal degradation. In conclusion, the down-modulation of CD45 expression in MCMV-infected myeloid cells represents a novel pathway of virus-host interaction., Author Summary Human cytomegalovirus (HCMV) is a tenacious pathogen, which can be life-threatening for immunocompromised patients and immunologically immature newborns. The pathogenicity of HCMV is owed to a plethora of immunomodulatory functions that interfere with host defense mechanisms. Such viral functions can teach us about viral pathogenesis mechanisms, and also about the functioning of immune cells. In this study we report that the mouse cytomegalovirus (MCMV)–a close relative of HCMV–influences surface expression of the cellular protein CD45 on macrophages and we identified the viral gene m42 mediating this effect. CD45 has long been known to be essential for the functioning of lymphocytes, however, its role in macrophages is less well understood. Growth analysis of a viral mutant indicated that the m42 gene confers a replication advantage to MCMV in vivo. We found that the m42 protein induces internalization of CD45 from the plasma membrane and degradation in lysosomes—most likely triggered by interaction of m42 with a ubiquitin ligase. In our study we detected a new element in the complex interaction of cytomegaloviruses with host cells, and further investigation into this mechanism may provide us with new insights into the functions of CD45 in myeloid cells.

Natural killer group 2 member D (NKG2D) is an activating receptor that is expressed on most cytotoxic cells of the immune system, including NK cells, γδ, and CD8

Cytomegalovirus (CMV) elicits long-term T-cell immunity of unparalleled strength, which has allowed the development of highly protective CMV-based vaccine vectors. Counterintuitively, experimental vaccines encoding a single MHC-I restricted epitope offered better immune protection than those expressing entire proteins, including the same epitope. To clarify this conundrum, we generated recombinant murine CMVs (MCMVs) encoding well-characterized MHC-I epitopes at different positions within viral genes and observed strong immune responses and protection against viruses and tumor growth when the epitopes were expressed at the protein C-terminus. We used the M45-encoded conventional epitope HGIRNASFI to dissect this phenomenon at the molecular level. A recombinant MCMV expressing HGIRNASFI on the C-terminus of M45, in contrast to wild-type MCMV, enabled peptide processing by the constitutive proteasome, direct antigen presentation, and an inflation of antigen-specific effector memory cells. Consequently, our results indicate that constitutive proteasome processing of antigenic epitopes in latently infected cells is required for robust inflationary responses. This insight allows utilizing the epitope positioning in the design of CMV-based vectors as a novel strategy for enhancing their efficacy., Author Summary Experimental cytomegalovirus (CMV) based vaccine vectors have provided highly encouraging results as innovative vaccine formulations against deadly virus infections, such as Ebola or AIDS. Nevertheless, it has remained incompletely understood why CMV is so efficient at stimulating T-lymphocytes, the immune cells that recognize pathogens within infected cells. We have generated an array of CMV mutants expressing the same antigen in different genes or in different parts of the same gene. This allowed us to identify that the immediate environment of the antigen, rather than properties of the antigen itself, crucially determine the immune protection conferred by CMV-based vaccines, implying that optimal immunity depends on the ability of host cells to degrade CMV proteins into peptides, short units that are recognized by T-cells. Detailed analysis revealed that strong and sustained T-cell immunity occurs only when their antigenic targets are processed by a primitive cellular machinery that is present in all cells of the body, rather than by its newly-evolved counterpart, which is present only in specialized antigen-presenting cells. Most importantly, our results provide a simple strategy to develop improved CMV vaccines by positioning the antigenic peptides at the right spot in CMV proteins.

With the cover headline 'T cells on the attack,' the journal Science celebrated individualized cancer immunotherapy by adoptive transfer of T cells as the 'Breakthrough of the Year' 2013 (J. Couzin-Frankel in Science 342:1432-1433, 2013). It is less well recognized and appreciated that individualized T cell immunotherapy of cytomegalovirus (CMV) infection is approaching clinical application for preventing CMV organ manifestations, interstitial CMV pneumonia in particular. This coincident medical development is particularly interesting as reactivated CMV infection is a major viral complication in the state of transient immunodeficiency after the therapy of hematopoietic malignancies by hematopoietic cell transplantation (HCT). It may thus be attractive to combine T cell immunotherapy of 'minimal residual disease/leukemia (MRD)' and CMV-specific T cell immunotherapy to combat both risks in HCT recipients simultaneously, and ideally with T cells derived from the respective HLA-matched HCT donor. Although clinical trials of human CMV-specific T cell immunotherapy were promising in that the incidence of virus reactivation and disease was found to be reduced with statistical significance, animal models are still instrumental for providing 'proof of concept' by directly documenting the prevention of viral multiple-organ histopathology and organ failure under controlled conditions of the absence versus presence of the therapy, which obviously is not feasible in an individual human patient. Further, animal models can make predictions regarding parameters that determine the efficacy of T cell immunotherapy for improved study design in clinical investigations, and they allow for manipulating host and virus genetics. The latter is of particular value as it opens the possibility for epitope specificity controls that are inherently missing in clinical trials. Here, we review a recently developed new mouse model that is more approximated to human CMV-specific T cell immunotherapy by 'humanizing' antigen presentation using antigenically chimeric CMV and HLA-transgenic mice to allow for an in vivo testing of the antiviral function of human CMV-specific T cells. As an important new message, this model predicts that T cell immunotherapy is most efficient if CD4 T cells are equipped with a transduced TCR directed against an epitope presented by MHC/HLA class-I for local delivery of 'cognate' help to CD8 effector T cells at infected MHC/HLA class-II-negative host tissue cells.

Cytomegalovirus (CMV) disease with multiple organ manifestations is the most feared viral complication limiting the success of hematopoietic cell transplantation as a therapy of hematopoietic malignancies. A timely endogenous reconstitution of CD8 T cells controls CMV infection, and adoptive transfer of antiviral CD8 T cells is a therapeutic option to prevent CMV disease by bridging the gap between an early CMV reactivation and delayed endogenous reconstitution of protective immunity. Preclinical research in murine models has provided 'proof of concept' for CD8 T-cell therapy of CMV disease. Protection by CD8 T cells appears to be in conflict with the finding that CMVs encode proteins that inhibit antigen presentation to CD8 T cells by interfering with the constitutive trafficking of peptide-loaded MHC class I molecules (pMHC-I complexes) to the cell surface. Here, we have systematically explored antigen presentation in the presence of the three currently noted immune evasion proteins of murine CMV in all possible combinations and its modulation by pre-treatment of cells with interferon-gamma (IFN-γ). The data reveal improvement in antigen processing by pre-treatment with IFN-γ can almost overrule the inhibitory function of immune evasion molecules in terms of pMHC-I expression levels capable of triggering most of the specific CD8 T cells, though the intensity of stimulation did not retrieve their full functional capacity. Notably, an in vivo conditioning of host tissue cells with IFN-γ in adoptive cell transfer recipients constitutively overexpressing IFN-γ (B6-SAP-IFN-γ mice) enhanced the antiviral efficiency of CD8 T cells in this transgenic cytoimmunotherapy model.

Medical interest in cytomegalovirus (CMV) is based on lifelong neurological sequelae, such as sensorineural hearing loss and mental retardation, resulting from congenital infection of the fetus in utero, as well as on CMV disease with multiple organ manifestations and graft loss in recipients of hematopoietic cell transplantation or solid organ transplantation. CMV infection of transplantation recipients occurs consequent to reactivation of virus harbored in a latent state in the transplanted donor cells and tissues, or in the tissues of the transplantation recipient herself or himself. Hence, CMV infection is a paradigm for a viral infection that causes disease primarily in the immunocompromised host, while infection of the immunocompetent host is associated with only mild and nonspecific symptoms so that it usually goes unnoticed. Thus, CMV is kept under strict immune surveillance. These medical facts are in apparent conflict with the notion that CMVs in general, human CMV as well as animal CMVs, are masters of 'immune evasion', which during virus-host co-speciation have convergently evolved sophisticated mechanisms to avoid their recognition by innate and adaptive immunity of their respective host species, with viral genes apparently dedicated to serve just this purpose (Reddehase in Nat Rev Immunol 2:831-844, 2002). With focus on viral interference with antigen presentation to CD8 T cells in the preclinical model of murine CMV infection, we try here to shed some more light on the in vivo balance between host immune surveillance of CMV infection and viral 'immune evasion' strategies.

The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted Gaussia luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by in vivo imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualize cells in which MCMV was being reactivated, precision-cut lung slices (PCLS) that preserve tissue microanatomy were prepared from the lungs of latently infected mice. By day 3 of cultivation of the PCLS, reactivation was revealed by Gluc expression, preceding the detection of infectious virus by approximately 4 days. Reactivation events in PCLS could be identified when they were still confined to single cells. Notably, using fractalkine receptor–GFP reporter mice, we never observed reactivation originating from CX3CR1+ monocytes or pulmonary dendritic cells derived therefrom. Furthermore, latent viral genome in the lungs was not enriched in sorted bone-marrow-derived cells expressing CD11b. Taken together, these complementary approaches suggest that CD11b+ and CX3CR1+ subsets of the myeloid differentiation lineage are not the main reservoirs and cellular sites of MCMV latency and reactivation in the lungs.

Cytomegaloviruses (CMVs) co-exist with their respective host species and have evolved to avoid their elimination by the hosts' immune effector mechanisms and to persist in a non-replicative state, known as viral latency. There is evidence to suggest that latency is nevertheless a highly dynamic condition during which episodes of viral gene desilencing, which can be viewed as incomplete reactivations, cause intermittent antigenic activity that stimulates CD8 memory-effector T cells and drives their clonal expansion. These T cells are supposed to terminate reactivation before completion of the productive viral cycle. In this view, CMVs do not "evade" their respective host's immune response but are actually held in check all the time, unless the host gets immunocompromised. Accordingly, CMV disease is typically a disease of the immunocompromised host only. Here we review current knowledge about the in vivo role of viral proteins involved in subverting the immune recognition of infected cells with focus on the CD8 T-cell response and viral interference with the MHC class-I pathway of antigenic peptide presentation. Whereas the intracellular functions of these "immune-evasion proteins" are known in molecular detail, knowledge of their in vivo role in CMV biology is only beginning to take shape. Experimental studies on the in vivo function of human CMV (hCMV) immune-evasion proteins prohibits, of course. Studying animal CMVs paradigmatically in the corresponding natural host is therefore used to identify principles from which the role of hCMV immune-evasion proteins can hopefully be inferred. Here we summarize recent insights gained primarily from the murine model.

Gene m164 of murine cytomegalovirus belongs to the large group of 'private' genes that show no homology to those of other cytomegalovirus species and are thought to represent 'host adaptation' genes involved in virus-host interaction. Previous interest in the m164 gene product was based on the presence of an immunodominant CD8 T-cell epitope presented at the surface of infected cells, despite interference by viral immune-evasion proteins. Here, we provide data to reveal that the m164 gene product shows unusual features in its cell biology. A novel strategy of mass-spectrometric analysis was employed to map the N terminus of the mature protein, 107 aa downstream of the start site of the predicted open reading frame. The resulting 36.5 kDa m164 gene product is identified here as an integral type-I membrane glycoprotein with exceptional intracellular trafficking dynamics, moving within the endoplasmic reticulum (ER) and outer nuclear membrane with an outstandingly high lateral membrane motility, actually 100 times higher than those published for cellular ER-resident proteins. Notably, gp36.5/m164 does not contain any typical ER-retention/retrieval signals, such as the C-terminal motifs KKXX or KXKXX, and does not pass the Golgi apparatus. Instead, it belongs to the rare group of viral glycoproteins in which the transmembrane domain (TMD) itself mediates direct ER retention. This is the first report relating TMD usage of an ER-resident transmembrane protein to its lateral membrane motility as a paradigm in cell biology. We propose that TMD usage for ER retention facilitates free and fast floating in ER-related membranes and between ER subdomains.

Preclinical research in murine models as well as subsequent clinical trials have concordantly revealed a high protective potential of antiviral CD8 T cells, of donor-derived ex vivo memory CD8 T cells in particular, in the immunotherapy of cytomegalovirus (CMV) infection in immunocompromised recipients. Although it is generally held view that the observed beneficial effect of the transferred cells is viral epitope-specific, involving the recognition of MHC class-I presented peptides by cognate T cell receptors, this assumption awaits formal proof, at least with regard to the in vivo function of the CD8 T cells. This question is particularly evident for CMV, since the function of viral immune evasion proteins interferes with the MHC class-I pathway of peptide presentation. Alternatively, therefore, one has to consider the possibility that the requirement for epitope recognition may be bypassed by other ligand-receptor interactions between CD8 T cells and infected cells, which may trigger the signaling for effector functions. Clearly, such a mechanism might explain why CD8 T cells are so efficient in controlling CMV infection despite the expression of viral immune evasion proteins. Here we provide direct evidence for epitope-specificity of antiviral protection by employing a recombinant murine CMV (mCMV), namely the mutant virus mCMV-IE1-L176A, in which an immunodominant viral epitope of the regulatory immediate-early protein IE1 is functionally deleted by a point mutation replacing leucine with alanine at the C-terminal MHC anchor position of the antigenic peptide.

Cytomegalovirus (CMV) is a ubiquitous virus, causing the most common congenital infection in humans, yet a vaccine against this virus is not available. Experimental studies of immunity against CMV in animal models of infection, such as the infection of mice with mouse CMV (MCMV), have relied mainly on parenteral infection protocols, although the virus naturally transmits by mucosal routes via body fluids. To characterize the biology of infections by mucosal routes, we compared the kinetics of virus replication, latent viral load and CD8 T-cell responses in lymphoid organs upon experimental intranasal (targeting the respiratory tract) and intragastric (targeting the digestive tract) infection with systemic intraperitoneal infection of two unrelated mouse strains. We observed that intranasal infection induced robust and long-term virus replication in the lungs and salivary glands but limited replication in the spleen. CD8 T-cell responses were somewhat weaker than upon intraperitoneal infection but showed similar kinetic profiles and phenotypes of antigen-specific cells. In contrast, intragastric infection resulted in abortive or poor virus replication in all tested organs and poor T-cell responses to the virus, especially at late times after infection. Consistent with the T-cell kinetics, the MCMV latent load was high in the lungs but low in the spleen of intranasally infected mice and lowest in all tested organs upon intragastric infection. In conclusion, we showed that intranasal but not intragastric infection of mice with MCMV represents a robust model to study the short- and long-term biology of CMV infection by a mucosal route.

Adoptive transfer of virus epitope-specific CD8 T cells is an immunotherapy option to control cytomegalovirus (CMV) infection and prevent CMV organ disease in immunocompromised solid organ transplantation (SOT) and hematopoietic cell transplantation (HCT) recipients. The therapy aims at an early, selective recognition and cytolysis of infected cells for preventing viral spread in tissues with no adverse immunopathogenic side-effects by attack of uninfected bystander cells. Here we describe that virus epitope-specific, cloned T-cell lines lyse target cells that present the cognate antigenic peptide to the TCR, but simultaneously have the potential to lyse uninfected cells expressing the CD28 ligand CD80 (B7-1). While TCR-mediated cytolysis requires co-receptor CD8 and depends on perforin, the TCR-independent and viral epitope-independent cytolysis through CD28–CD80 signaling does not require CD8 on the effector cells and is perforin-independent. Importantly, this non-cognate cytolysis pathway leads to bystander cytolysis of CD80-expressing B-cell blasts and thereby inhibits pan-specific antibody production.

Reactivation of human cytomegalovirus (HCMV) can cause severe disease in recipients of hematopoietic stem cell transplantation. Although preclinical research in murine models as well as clinical trials have provided 'proof of concept' for infection control by pre-emptive CD8 T-cell immunotherapy, there exists no predictive model to experimentally evaluate parameters that determine antiviral efficacy of human T cells in terms of virus control in functional organs, prevention of organ disease, and host survival benefit. We here introduce a novel mouse model for testing HCMV epitope-specific human T cells. The HCMV UL83/pp65-derived NLV-peptide was presented by transgenic HLA-A2.1 in the context of a lethal infection of NOD/SCID/IL-2rg-/- mice with a chimeric murine CMV, mCMV-NLV. Scenarios of HCMV-seropositive and -seronegative human T-cell donors were modeled by testing peptide-restimulated and T-cell receptor-transduced human T cells, respectively. Upon transfer, the T cells infiltrated host tissues in an epitope-specific manner, confining the infection to nodular inflammatory foci. This resulted in a significant reduction of viral load, diminished organ pathology, and prolonged survival. The model has thus proven its potential for a preclinical testing of the protective antiviral efficacy of HCMV epitope-specific human T cells in the evaluation of new approaches to an immunotherapy of CMV disease., Author Summary Pre-emptive CD8 T-cell therapy of human cytomegalovirus (HCMV) disease in immunocompromised recipients of hematopoietic stem cell transplantation gave promising results in clinical trials, but limited efficacy and the need of HCMV-seropositive memory cell donors has so far prevented adoptive cell transfer from becoming clinical routine. Further development is currently hampered by the lack of experimental animal models that allow preclinical testing of the protective efficacy of human T cells in functional organs. While humanized mouse models with human tissue implants are technically and statistically demanding, and are limited to studying human T-cell activation and local virus control in the implants, a more feasible model for control of systemic infection and prevention of multiple-organ CMV disease is regrettably missing. Here we introduce such a model based on infection of genetically immunocompromised, HLA-A2.1-transgenic NOD/SCID/IL-2rg-/- mice with a chimeric murine CMV engineered to express the HCMV NLV-peptide epitope. Mimicking the scenario of HCMV-unexperienced donors, human T cells transduced with a human T-cell receptor specific for HLA-A.2.1-presented NLV peptide controlled systemic infection and moderated organ disease resulting in a survival benefit. The model promises to become instrumental in defining T-cell properties that determine their protective efficacy for a further development of adoptive immunotherapy of post-transplantation CMV infection.

Viruses have evolved proteins that bind immunologically relevant cargo molecules at the cell surface for their downmodulation by internalization. Via a tyrosine-based sorting motif YXXΦ in their cytoplasmic tails, they link the bound cargo to the cellular adapter protein-2 (AP2), thereby sorting it into clathrin-triskelion-coated pits for accelerated endocytosis. Downmodulation of CD4 molecules by lentiviral protein NEF represents the most prominent example. Based on connecting cargo to cellular adapter molecules, such specialized viral proteins have been referred to as ‘connectors’ or ‘adapter adapters.’ Murine cytomegalovirus glycoprotein m04/gp34 binds stably to MHC class-I (MHC-I) molecules and suspiciously carries a canonical YXXΦ endocytosis motif YRRF in its cytoplasmic tail. Disconnection from AP2 by motif mutation ARRF should retain m04-MHC-I complexes at the cell surface and result in an enhanced silencing of natural killer (NK) cells, which recognize them via inhibitory receptors. We have tested this prediction with a recombinant virus in which the AP2 motif is selectively destroyed by point mutation Y248A, and compared this with the deletion of the complete protein in a Δm04 mutant. Phenotypes were antithetical in that loss of AP2-binding enhanced NK cell silencing, whereas absence of m04-MHC-I released them from silencing. We thus conclude that AP2-binding antagonizes NK cell silencing by enhancing endocytosis of the inhibitory ligand m04-MHC-I. Based on a screen for tyrosine-based endocytic motifs in cytoplasmic tail sequences, we propose here the new hypothesis that most proteins of the m02–m16 gene family serve as ‘adapter adapters,’ each selecting its specific cell surface cargo for clathrin-assisted internalization.

Control of murine cytomegalovirus (mCMV) infection is mediated primarily by CD8 T cells, with four specificities dominating in BALB/c mice. Functional deletion of the respective immunodominant epitopes (IDEs) in mutant virus Δ4IDE revealed a still efficient control of infection. In a murine model of hematopoietic cell transplantation and infection with Δ4IDE, an mCMV-specific open reading frame (ORF) library screening assay indicated a strong CD8 T cell reactivity against the ORF-M54 product, the highly conserved and essential mCMV homolog of human CMV DNA polymerase UL54, which is a known inducer of in vivo protection against mCMV by DNA immunization. Applying bioinformatic algorithms for CD8 T cell epitope prediction, the top-scoring peptides were used to stimulate ex vivo-isolated CD8 T cells and to generate cytolytic T cell lines; yet, this approach failed to identify M54 epitope(s). As an alternative, a peptide library consisting of 549 10-mers with an offset of two amino acids (aa), covering the complete aa-sequence of the M54 protein, was synthesized and used for the stimulation. A region of 12 aa proved to encompass an epitope. An 'alanine walk' over this antigenic 12-mer and all possible 11-, 10- and 9-mers derived thereof revealed aa-residues critical for antigenicity, and terminal truncations identified the H-2D(d) presented 8-mer M5483-90 as the optimal epitope. An increased frequency of the corresponding CD8 T cells in the absence of the 4 IDEs indicated immunodomination by the IDE-specific CD8 T cells as a mechanism by which the generation of M54-specific CD8 T cells is inhibited after infection with wild-type mCMV.

Herpesviruses form different gH/gL virion envelope glycoprotein complexes that serve as entry complexes for mediating viral cell-type tropism in vitro; their roles in vivo, however, remained speculative and can be addressed experimentally only in animal models. For murine cytomegalovirus two alternative gH/gL complexes, gH/gL/gO and gH/gL/MCK-2, have been identified. A limitation of studies on viral tropism in vivo has been the difficulty in distinguishing between infection initiation by viral entry into first-hit target cells and subsequent cell-to-cell spread within tissues. As a new strategy to dissect these two events, we used a gO-transcomplemented ΔgO mutant for providing the gH/gL/gO complex selectively for the initial entry step, while progeny virions lack gO in subsequent rounds of infection. Whereas gH/gL/gO proved to be critical for establishing infection by efficient entry into diverse cell types, including liver macrophages, endothelial cells, and hepatocytes, it was dispensable for intra-tissue spread. Notably, the salivary glands, the source of virus for host-to-host transmission, represent an exception in that entry into virus-producing cells did not strictly depend on either the gH/gL/gO or the gH/gL/MCK-2 complex. Only if both complexes were absent in gO and MCK-2 double-knockout virus, in vivo infection was abolished at all sites., Author Summary The role of viral glycoprotein entry complexes in viral tropism in vivo is a question central to understanding virus pathogenesis and transmission for any virus. Studies were limited by the difficulty in distinguishing between viral entry into first-hit target cells and subsequent cell-to-cell spread within tissues. Employing the murine cytomegalovirus entry complex gH/gL/gO as a paradigm for a generally applicable strategy to dissect these two events experimentally, we used a gO-transcomplemented ΔgO mutant for providing the complex exclusively for the initial cell entry step. In immunocompromised mice as a model for recipients of hematopoietic cell transplantation, our studies revealed an irreplaceable role for gH/gL/gO in initiating infection in host organs relevant to pathogenesis, whereas subsequent spread within tissues and infection of the salivary glands, the site relevant to virus host-to-host transmission, are double-secured by the entry complexes gH/gL/gO and gH/gL/MCK-2. As an important consequence, interventional strategies targeting only gO might be efficient in preventing organ manifestations after a primary viremia, whereas both gH/gL complexes need to be targeted for preventing intra-tissue spread of virus reactivated from latency within tissues as well as for preventing the salivary gland route of host-to-host transmission.

Reactivation of latent cytomegalovirus (CMV) in the transient immunocompromised state after hematoablative treatment is a major concern in patients undergoing hematopoietic cell transplantation (HCT) as a therapy of hematopoietic malignancies. Timely reconstitution of antiviral CD8 T cells and their efficient recruitment to the lungs is crucial for preventing interstitial pneumonia, the most severe disease manifestation of CMV in HCT recipients. Here, we review recent work in a murine model, implicating mast cells (MC) in the control of pulmonary infection. Murine CMV (mCMV) productively infects MC in vivo and triggers their degranulation, resulting in the release of the CC chemokine ligand 5 (CCL5) that attracts CD8 T cells to infiltrate infected tissues. Comparing infection of MC-sufficient C57BL/6 mice and congenic MC-deficient Kit W-sh/W-sh “sash” mutants revealed an inverse relation between the number of lung-infiltrating CD8 T cells and viral burden in the lungs. Specifically, reduced lung infiltration by CD8 T cells in “sash” mutants was associated with an impaired infection control. The causal, though indirect, involvement of MC in antiviral control was confirmed by reversion of the deficiency phenotype in “sash” mutants reconstituted with MC. These recent findings predict that efficient MC reconstitution facilitates the control of CMV infection also in immunocompromised HCT recipients.

The succinct metaphor, ‘the immune system's loaded gun', has been used to describe the role of mast cells (MCs) due to their storage of a wide range of potent pro-inflammatory and antimicrobial mediators in secretory granules that can be released almost instantly on demand to fight invaders. Located at host–environment boundaries and equipped with an arsenal of pattern recognition receptors, MCs are destined to be rapid innate sensors of pathogens penetrating endothelial and epithelial surfaces. Although the importance of MCs in antimicrobial and antiparasitic defense has long been appreciated, their role in raising the alarm against viral infections has been noted only recently. Work on cytomegalovirus (CMV) infection in the murine model has revealed MCs as players in a novel cross-talk axis between innate and adaptive immune surveillance of CMV, in that infection of MCs, which is associated with MC degranulation and release of the chemokine CCL5, enhances the recruitment of protective CD8 T cells to extravascular sites of virus replication, specifically to lung interstitium and alveolar epithelium. Here, we have expanded on these studies by investigating the conditions for MC activation and the consequent degranulation in response to host infection. Surprisingly, the data revealed two temporally and mechanistically distinct waves of MC activation: an almost instant indirect activation that depended on TLR3/TRIF signaling and delayed activation by direct infection of MCs that did not involve TLR3/TRIF signaling. Cell type-specific Cre-recombination that yielded eGFP-expressing reporter virus selectively originating from MCs identified MC as a new in vivo, first-hit target cell of productive murine CMV infection.