The activation trajectory of plasmacytoid dendritic cells in vivo during a viral infection.

Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN-I). What other functions pDCs exert in vivo during viral infections is controversial, and more studies are needed to understand their orchestration. In the present study, we characterize in depth and link pDC activation states in animals infected by mouse cytomegalovirus by combining Ifnb1 reporter mice with flow cytometry, single-cell RNA sequencing, confocal microscopy and a cognate CD4 T cell activation assay. We show that IFN-I production and T cell activation were performed by the same pDC, but these occurred sequentially in time and in different micro-anatomical locations. In addition, we show that pDC commitment to IFN-I production was marked early on by their downregulation of leukemia inhibitory factor receptor and was promoted by cell-intrinsic tumor necrosis factor signaling. We propose a new model for how individual pDCs are endowed to exert different functions in vivo during a viral infection, in a manner tightly orchestrated in time and space.

ILC1 Confer Early Host Protection at Initial Sites of Viral Infection.

Infection is restrained by the concerted activation of tissue-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.

Syndromic immune disorder caused by a viable hypomorphic allele of spliceosome component Snrnp40.

We report a new immunodeficiency disorder in mice caused by a viable hypomorphic mutation of Snrnp40, an essential gene encoding a subunit of the U5 small nuclear ribonucleoprotein (snRNP) complex of the spliceosome. Snrnp40 is ubiquitous but strongly expressed in lymphoid tissue. Homozygous mutant mice showed hypersusceptibility to infection by murine cytomegalovirus and multiple defects of lymphoid development, stability and function. Cell-intrinsic defects of hematopoietic stem cell differentiation also affected homozygous mutants. SNRNP40 deficiency in primary hematopoietic stem cells or T cells or the EL4 cell line increased the frequency of splicing errors, mostly intron retention, in several hundred messenger RNAs. Altered expression of proteins associated with immune cell function was also observed in Snrnp40-mutant cells. The immunological consequences of SNRNP40 deficiency presumably result from cumulative, moderate effects on processing of many different mRNA molecules and secondary reductions in the expression of critical immune proteins, yielding a syndromic immune disorder.

Epigenetic control of innate and adaptive immune memory.

Clonal expansion and immunological memory are hallmark features of the mammalian adaptive immune response and essential for prolonged host control of pathogens. Recent work demonstrates that natural killer (NK) cells of the innate immune system also exhibit these adaptive traits during infection. Here we demonstrate that differentiating and 'memory' NK cells possess distinct chromatin accessibility states and that their epigenetic profiles reveal a 'poised' regulatory program at the memory stage. Furthermore, we elucidate how individual STAT transcription factors differentially control epigenetic and transcriptional states early during infection. Finally, concurrent chromatin profiling of the canonical CD8+ T cell response against the same infection demonstrated parallel and distinct epigenetic signatures defining NK cells and CD8+ T cells. Overall, our study reveals the dynamic nature of epigenetic modifications during the generation of innate and adaptive lymphocyte memory.

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 after infection with mouse cytomegalovirus (MCMV). Mechanistically, endogenous glucocorticoids produced shortly after infection induced selective and tissue-specific expression of the checkpoint receptor PD-1 on NK cells. This glucocorticoid-PD-1 pathway limited production of the cytokine IFN-γ by spleen NK cells, which prevented immunopathology. Notably, 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.

The RAG recombinase dictates functional heterogeneity and cellular fitness in natural killer cells.

The emergence of recombination-activating genes (RAGs) in jawed vertebrates endowed adaptive immune cells with the ability to assemble a diverse set of antigen receptor genes. In contrast, innate lymphocytes, such as natural killer (NK) cells, are not believed to require RAGs. Here, we report that NK cells unable to express RAGs or RAG endonuclease activity during ontogeny exhibit a cell-intrinsic hyperresponsiveness but a diminished capacity to survive following virus-driven proliferation, a reduced expression of DNA damage response mediators, and defects in the repair of DNA breaks. Evidence for this novel function of RAG has also been observed in T cells and innate lymphoid cells (ILCs), revealing an unexpected role for RAG proteins beyond V(D)J recombination. We propose that DNA cleavage events mediated by RAG endow developing adaptive and innate lymphocytes with a cellular "fitness" that safeguards their persistence later in life during episodes of rapid proliferation or cellular stress.

NFAT signaling is indispensable for persistent memory responses of MCMV-specific CD8+ T cells.

Cytomegalovirus (CMV) induces a unique T cell response, where antigen-specific populations do not contract, but rather inflate during viral latency. It has been proposed that subclinical episodes of virus reactivation feed the inflation of CMV-specific memory cells by intermittently engaging T cell receptors (TCRs), but evidence of TCR engagement has remained lacking. Nuclear factor of activated T cells (NFAT) is a family of transcription factors, where NFATc1 and NFATc2 signal downstream of TCR in mature T lymphocytes. We show selective impacts of NFATc1 and/or NFATc2 genetic ablations on the long-term inflation of MCMV-specific CD8+ T cell responses despite largely maintained responses to acute infection. NFATc1 ablation elicited robust phenotypes in isolation, but the strongest effects were observed when both NFAT genes were missing. CMV control was impaired only when both NFATs were deleted in CD8+ T cells used in adoptive immunotherapy of immunodeficient mice. Transcriptome analyses revealed that T cell intrinsic NFAT is not necessary for CD8+ T cell priming, but rather for their maturation towards effector-memory and in particular the effector cells, which dominate the pool of inflationary cells.

Spatial kinetics and immune control of murine cytomegalovirus infection in the salivary glands.

Human cytomegalovirus (HCMV) is the most common congenital infection. Several HCMV vaccines are in development, but none have yet been approved. An understanding of the kinetics of CMV replication and transmission may inform the rational design of vaccines to prevent this infection. The salivary glands (SG) are an important site of sustained CMV replication following primary infection and during viral reactivation from latency. As such, the strength of the immune response in the SG likely influences viral dissemination within and between hosts. To study the relationship between the immune response and viral replication in the SG, and viral dissemination from the SG to other tissues, mice were infected with low doses of murine CMV (MCMV). Following intra-SG inoculation, we characterized the viral and immunological dynamics in the SG, blood, and spleen, and identified organ-specific immune correlates of protection. Using these data, we constructed compartmental mathematical models of MCMV infection. Model fitting to data and analysis indicate the importance of cellular immune responses in different organs and point to a threshold of infection within the SG necessary for the establishment and spread of infection.

Mouse cytomegalovirus lacking sgg1 shows reduced import into the salivary glands.

Cytomegaloviruses (CMVs) transmit via chronic shedding from the salivary glands. How this relates to the broad cell tropism they exhibit in vitro is unclear. Human CMV (HCMV) infection presents only after salivary gland infection is established. Murine CMV (MCMV) is therefore useful to analyse early infection events. It reaches the salivary glands via infected myeloid cells. Three adjacent spliced genes designated as m131/129 (MCK-2), sgg1 and sgg1.1, positional homologues of the HCMV UL128/130/131 tropism determinants, are implicated. We show that a sgg1 null mutant is defective in infected myeloid cell entry into the salivary glands, a phenotype distinct from MCMV lacking MCK-2. These data point to a complex, multi-step process of salivary gland colonization.

Influence of Self-MHC Class I Recognition on the Dynamics of NK Cell Responses to Cytomegalovirus Infection.

Although interactions between inhibitory Ly49 receptors and their self-MHC class I ligands in C57BL/6 mice are known to limit NK cell proliferation during mouse CMV (MCMV) infection, we created a 36-marker mass cytometry (CyTOF) panel to investigate how these inhibitory receptors impact the NK cell response to MCMV in other phenotypically measurable ways. More than two thirds of licensed NK cells (i.e., those expressing Ly49C, Ly49I, or both) in uninfected mice had already differentiated into NK cells with phenotypes indicative of Ag encounter (KLRG1+Ly6C-) or memory-like status (KLRG1+Ly6C+). These pre-existing KLRG1+Ly6C+ NK cells resembled known Ag-specific memory NK cell populations in being less responsive to IL-18 and IFN-α stimulation in vitro and by selecting for NK cell clones with elevated expression of a Ly49 receptor. During MCMV infection, the significant differences between licensed and unlicensed (Ly49C-Ly49I-) NK cells disappeared within both CMV-specific (Ly49H+) and nonspecific (Ly49H-) responses. This lack of heterogeneity carried into the memory phase, with only a difference in CD16 expression manifesting between licensed and unlicensed MCMV-specific memory NK cell populations. Our results suggest that restricting proliferation is the predominant effect licensing has on the NK cell population during MCMV infection, but the inhibitory Ly49-MHC interactions that take place ahead of infection contribute to their limited expansion by shrinking the pool of licensed NK cells capable of robustly responding to new challenges.

Characterization of M116.1p, a Murine Cytomegalovirus Protein Required for Efficient Infection of Mononuclear Phagocytes.

Broad tissue tropism of cytomegaloviruses (CMVs) is facilitated by different glycoprotein entry complexes, which are conserved between human CMV (HCMV) and murine CMV (MCMV). Among the wide array of cell types susceptible to the infection, mononuclear phagocytes (MNPs) play a unique role in the pathogenesis of the infection as they contribute both to the virus spread and immune control. CMVs have dedicated numerous genes for the efficient infection and evasion of macrophages and dendritic cells. In this study, we have characterized the properties and function of M116, a previously poorly described but highly transcribed MCMV gene region that encodes M116.1p, a novel protein necessary for the efficient infection of MNPs and viral spread in vivo. Our study further revealed that M116.1p shares similarities with its positional homologs in HCMV and RCMV, UL116 and R116, respectively, such as late kinetics of expression, N-glycosylation, localization to the virion assembly compartment, and interaction with gH-a member of the CMVs fusion complex. This study, therefore, expands our knowledge about virally encoded glycoproteins that play important roles in viral infectivity and tropism. IMPORTANCE Human cytomegalovirus (HCMV) is a species-specific herpesvirus that causes severe disease in immunocompromised individuals and immunologically immature neonates. Murine cytomegalovirus (MCMV) is biologically similar to HCMV, and it serves as a widely used model for studying the infection, pathogenesis, and immune responses to HCMV. In our previous work, we have identified the M116 ORF as one of the most extensively transcribed regions of the MCMV genome without an assigned function. This study shows that the M116 locus codes for a novel protein, M116.1p, which shares similarities with UL116 and R116 in HCMV and RCMV, respectively, and is required for the efficient infection of mononuclear phagocytes and virus spread in vivo. Furthermore, this study establishes the α-M116 monoclonal antibody and MCMV mutants lacking M116, generated in this work, as valuable tools for studying the role of macrophages and dendritic cells in limiting CMV infection following different MCMV administration routes.

The Mouse Cytomegalovirus G Protein-Coupled Receptor Homolog, M33, Coordinates Key Features of In Vivo Infection via Distinct Components of Its Signaling Repertoire.

Common to all cytomegalovirus (CMV) genomes analyzed to date is the presence of G protein-coupled receptors (GPCR). Animal models of CMV provide insights into their role in viral fitness. The mouse cytomegalovirus (MCMV) GPCR, M33, facilitates dendritic cell (DC)-dependent viremia, the extravasation of blood-borne infected DCs to the salivary gland, and the frequency of reactivation events from latently infected tissue explants. Constitutive G protein-coupled M33 signaling is required for these phenotypes, although the contribution of distinct biochemical pathways activated by M33 is unknown. M33 engages Gq/11 to constitutively activate phospholipase C ß (PLCß) and downstream cyclic AMP response-element binding protein (CREB) in vitro. Identification of a MCMV M33 mutant (M33ΔC38) for which CREB signaling was disabled but PLCß activation was preserved provided the opportunity to investigate their relevance in vivo. Following intranasal infection with MCMV M33ΔC38, the absence of M33 CREB Gq/11-dependent signaling correlated with reduced mobilization of lytically-infected DCs to the draining lymph node high endothelial venules (HEVs) and reduced viremia compared with wild type MCMV. In contrast, M33ΔC38-infected DCs within the vascular compartment extravasated to the salivary glands via a pertussis toxin-sensitive, Gi/o-dependent, and CREB-independent mechanism. In the context of MCMV latency, spleen explants from M33ΔC38-infected mice were markedly attenuated for reactivation. Taken together, these data demonstrate that key features of the MCMV life cycle are coordinated in diverse tissues by distinct pathways of the M33 signaling repertoire. IMPORTANCE G protein-coupled receptors (GPCRs) act as cell surface molecular "switches" that regulate the cellular response to environmental stimuli. All cytomegalovirus (CMV) genomes analyzed to date possess GPCR homologs with phylogenetic evidence for independent gene capture events, signifying important in vivo roles. The mouse CMV (MCMV) GPCR homolog, designated M33, is important for cell-associated virus spread and the establishment and/or reactivation of latent MCMV infection. The signaling repertoire of M33 is distinct from cellular GPCRs and little is known of the relevance of component signaling pathways for in vivo M33 function. In this report, we showed that temporal and tissue-specific M33 signaling was required to facilitate in vivo infection. Understanding the relevance of the viral GPCR signaling profiles for in vivo function will provide opportunities for future targeted interventions.

Hematopoietic cell-mediated dissemination of murine cytomegalovirus is regulated by NK cells and immune evasion.

Cytomegalovirus (CMV) causes clinically important diseases in immune compromised and immune immature individuals. Based largely on work in the mouse model of murine (M)CMV, there is a consensus that myeloid cells are important for disseminating CMV from the site of infection. In theory, such dissemination should expose CMV to cell-mediated immunity and thus necessitate evasion of T cells and NK cells. However, this hypothesis remains untested. We constructed a recombinant MCMV encoding target sites for the hematopoietic specific miRNA miR-142-3p in the essential viral gene IE3. This virus disseminated poorly to the salivary gland following intranasal or footpad infections but not following intraperitoneal infection in C57BL/6 mice, demonstrating that dissemination by hematopoietic cells is essential for specific routes of infection. Remarkably, depletion of NK cells or T cells restored dissemination of this virus in C57BL/6 mice after intranasal infection, while dissemination occurred normally in BALB/c mice, which lack strong NK cell control of MCMV. These data show that cell-mediated immunity is responsible for restricting MCMV to hematopoietic cell-mediated dissemination. Infected hematopoietic cells avoided cell-mediated immunity via three immune evasion genes that modulate class I MHC and NKG2D ligands (m04, m06 and m152). MCMV lacking these 3 genes spread poorly to the salivary gland unless NK cells were depleted, but also failed to replicate persistently in either the nasal mucosa or salivary gland unless CD8+ T cells were depleted. Surprisingly, CD8+ T cells primed after intranasal infection required CD4+ T cell help to expand and become functional. Together, our data suggest that MCMV can use both hematopoietic cell-dependent and -independent means of dissemination after intranasal infection and that cell mediated immune responses restrict dissemination to infected hematopoietic cells, which are protected from NK cells during dissemination by viral immune evasion. In contrast, viral replication within mucosal tissues depends on evasion of T cells.

Long-Term Evolution of the Adaptive NKG2C+ NK Cell Response to Cytomegalovirus Infection in Kidney Transplantation: An Insight on the Diversity of Host-Pathogen Interaction.

Human CMV infection is frequent in kidney transplant recipients (KTR). Pretransplant Ag-specific T cells and adaptive NKG2C+ NK cells associate with reduced incidence of infection in CMV+ KTR. Expansions of adaptive NKG2C+ NK cells were reported in posttransplant CMV-infected KTR. To further explore this issue, NKG2C+ NK, CD8+, and TcRγδ T cells were analyzed pretransplant and at different time points posttransplant for ≥24 mo in a cohort of CMV+ KTR (n = 112), stratified according to CMV viremia detection. In cryopreserved samples from a subgroup (n = 49), adaptive NKG2C+ NK cell markers and T cell subsets were compared after a longer follow-up (median, 56 mo), assessing the frequencies of CMV-specific T cells and viremia at the last time point. Increased proportions of NKG2C+ NK, CD8+, and TcRγδ T cells were detected along posttransplant evolution in viremia(+) KTR. However, the individual magnitude and kinetics of the NKG2C+ NK response was variable and only exceptionally detected among viremia(-) KTR, presumably reflecting subclinical viral replication events. NKG2C+ expansions were independent of KLRC2 zygosity and associated with higher viral loads at diagnosis; no relation with other clinical parameters was perceived. Increased proportions of adaptive NKG2C+ NK cells (CD57+, ILT2+, FcεRIγ-) were observed after resolution of viremia long-term posttransplant, coinciding with increased CD8+ and Vδ2- γδ T cells; at that stage CMV-specific T cells were comparable to viremia(-) cases. These data suggest that adaptive NKG2C+ NK cells participate with T cells to restore CMV replication control, although their relative contribution cannot be discerned.

Transcriptome Analysis of Retinal and Choroidal Pathologies in Aged BALB/c Mice Following Systemic Neonatal Murine Cytomegalovirus Infection.

Our previous studies have shown that systemic neonatal murine cytomegalovirus (MCMV) infection of BALB/c mice spread to the eye with subsequent establishment of latency in choroid/RPE. In this study, RNA sequencing (RNA-Seq) analysis was used to determine the molecular genetic changes and pathways affected by ocular MCMV latency. MCMV (50 pfu per mouse) or medium as control were injected intra-peritoneally (i.p.) into BALB/c mice at <3 days after birth. At 18 months post injection, the mice were euthanized, and the eyes were collected and prepared for RNA-Seq. Compared to three uninfected control eyes, we identified 321 differentially expressed genes (DEGs) in six infected eyes. Using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA), we identified 17 affected canonical pathways, 10 of which function in neuroretinal signaling, with the majority of DEGs being downregulated, while 7 pathways function in upregulated immune/inflammatory responses. Retinal and epithelial cell death pathways involving both apoptosis and necroptosis were also activated. MCMV ocular latency is associated with upregulation of immune and inflammatory responses and downregulation of multiple neuroretinal signaling pathways. Cell death signaling pathways are also activated and contribute to the degeneration of photoreceptors, RPE, and choroidal capillaries.

Docosahexaenoic acid supplementation represses the early immune response against murine cytomegalovirus but enhances NK cell effector function.

BACKGROUND: Docosahexaenoic acid (DHA) supplementation is beneficial for several chronic diseases; however, its effect on immune regulation is still debated. Given the prevalence of cytomegalovirus (CMV) infection and because natural killer (NK) cells are a component of innate immunity critical for controlling CMV infection, the current study explored the effect of a DHA-enriched diet on susceptibility to murine (M) CMV infection and the NK cell effector response to MCMV infection. RESULTS: Male C57BL/6 mice fed a control or DHA-enriched diet for 3 weeks were infected with MCMV and sacrificed at the indicated time points postinfection. Compared with control mice, DHA-fed mice had higher liver and spleen viral loads at day 7 postinfection, but final MCMV clearance was not affected. The total numbers of NK cells and their terminal mature cell subset (KLRG1+ and Ly49H+ NK cells) were reduced compared with those in control mice at day 7 postinfection but not day 21. DHA feeding resulted in higher IFN-γ and granzyme B expression in splenic NK cells at day 7 postinfection. A mechanistic analysis showed that the splenic NK cells of DHA-fed mice had enhanced glucose uptake, increased CD71 and CD98 expression, and higher mitochondrial mass than control mice. In addition, DHA-fed mice showed reductions in the total numbers and activation levels of CD4+ and CD8+ T cells. CONCLUSIONS: These results suggest that DHA supplementation represses the early response to CMV infection but preserves NK cell effector functions by improving mitochondrial activity, which may play critical roles in subsequent MCMV clearance.

PD-1-specific "Blocking" antibodies that deplete PD-1+ T cells present an inconvenient variable in preclinical immunotherapy experiments.

Therapeutic antibodies blocking PD-1-/PD-L1 interaction have achieved remarkable clinical success in cancer. In addition to blocking a target molecule, some isotypes of antibodies can activate complement, NK cells or phagocytes, resulting in death of the cell expressing the antibody's target. Human anti-PD-1 therapeutics use antibody isotypes designed to minimize such antibody-dependent lysis. In contrast, anti-PD-1 reagents used in mice are derived from multiple species, with different isotypes, and are not engineered to reduce target cell death: few studies analyze or discuss how antibody species and isotype may impact data interpretation. We demonstrate here that anti-PD-1 therapy to promote activation and proliferation of murine PD-1-expressing CD8 T cells sometimes led instead to a loss of antigen specific cells. This phenomenon was seen in two tumor models and a model of virus infection, and varied with the clone of anti-PD-1 antibody. Additionally, we compared competition among anti-PD-1 clones to find a combination that allows detection of PD-1-expressing cells despite the presence of blocking anti-PD1 antibodies in vivo. These data bring attention to the possibility of unintended target cell depletion with some commonly used anti-mouse PD-1 clones, and should provide a valuable resource for the design and interpretation of anti-PD-1 studies in mice.

Murine Cytomegalovirus MCK-2 Facilitates In Vivo Infection Transfer from Dendritic Cells to Salivary Gland Acinar Cells.

The cytomegaloviruses (CMVs) spread systemically via myeloid cells and demonstrate broad tissue tropism. Human CMV (HCMV) UL128 encodes a component of the virion pentameric complex (PC) that is important for entry into epithelial cells and cell-cell spread in vitro. It possesses N-terminal amino acid sequences similar to those of CC chemokines. While the species specificity of HCMV precludes confirmation of UL128 function in vivo, UL128-like counterparts in experimental animals have demonstrated a role in salivary gland infection. How they achieve this has not been defined, although effects on monocyte tropism and immune evasion have been proposed. By tracking infected cells following lung infection, we show that although the UL128-like protein in mouse CMV (MCMV) (designated MCK-2) facilitated entry into lung macrophages, it was dispensable for subsequent viremia mediated by CD11c+ dendritic cells (DCs) and extravasation to the salivary glands. Notably, MCK-2 was important for the transfer of MCMV infection from DCs to salivary gland acinar epithelial cells. Acinar cell infection of MCMVs deleted of MCK-2 was not rescued by T-cell depletion, arguing against an immune evasion mechanism for MCK-2 in the salivary glands. In contrast to lung infection, peritoneal MCMV inoculation yields mixed monocyte/DC viremia. In this setting, MCK-2 again promoted DC-dependent infection of salivary gland acinar cells, but it was not required for monocyte-dependent spread to the lung. Thus, the action of MCK-2 in MCMV spread was specific to DC-acinar cell interactions. IMPORTANCE Cytomegaloviruses (CMVs) establish myeloid cell-associated viremias and persistent shedding from the salivary glands. In vitro studies with human CMV (HCMV) have implicated HCMV UL128 in epithelial tropism, but its role in vivo is unknown. Here, we analyzed how a murine CMV (MCMV) protein with similar physical properties, designated MCK-2, contributes to host colonization. We demonstrate that MCK-2 is dispensable for initial systemic spread from primary infection sites but within the salivary gland facilitates the transfer of infection from dendritic cells (DCs) to epithelial acinar cells. Virus transfer from extravasated monocytes to the lungs did not require MCK-2, indicating a tissue-specific effect. These results provide new information about how persistent viral tropism determinants operate in vivo.

Murine cytomegaloviruses m139 targets DDX3 to curtail interferon production and promote viral replication.

Cytomegaloviruses (CMV) infect many different cell types and tissues in their respective hosts. Monocytes and macrophages play an important role in CMV dissemination from the site of infection to target organs. Moreover, macrophages are specialized in pathogen sensing and respond to infection by secreting cytokines and interferons. In murine cytomegalovirus (MCMV), a model for human cytomegalovirus, several genes required for efficient replication in macrophages have been identified, but their specific functions remain poorly understood. Here we show that MCMV m139, a gene of the conserved US22 gene family, encodes a protein that interacts with the DEAD box helicase DDX3, a protein involved in pathogen sensing and interferon (IFN) induction, and the E3 ubiquitin ligase UBR5. DDX3 and UBR5 also participate in the transcription, processing, and translation of a subset of cellular mRNAs. We show that m139 inhibits DDX3-mediated IFN-α and IFN-ß induction and is necessary for efficient viral replication in bone-marrow derived macrophages. In vivo, m139 is crucial for viral dissemination to local lymph nodes and to the salivary glands. An m139-deficient MCMV also replicated to lower titers in SVEC4-10 endothelial cells. This replication defect was not accompanied by increased IFN-ß transcription, but was rescued by knockout of either DDX3 or UBR5. Moreover, m139 co-localized with DDX3 and UBR5 in viral replication compartments in the cell nucleus. These results suggest that m139 inhibits DDX3-mediated IFN production in macrophages and antagonizes DDX3 and UBR5-dependent functions related to RNA metabolism in endothelial cells.

Retinal and Choroidal Pathologies in Aged BALB/c Mice Following Systemic Neonatal Murine Cytomegalovirus Infection.

Although pathologies associated with acute virus infections have been extensively studied, the effects of long-term latent virus infections are less well understood. Human cytomegalovirus, which infects 50% to 80% of humans, is usually acquired during early life and persists in a latent state for the lifetime. The purpose of this study was to determine whether systemic murine cytomegalovirus (MCMV) infection acquired early in life disseminates to and becomes latent in the eye and if ocular MCMV can trigger in situ inflammation and occurrence of ocular pathology. This study found that neonatal infection of BALB/c mice with MCMV resulted in dissemination of virus to the eye, where it localized principally to choroidal endothelia and pericytes and less frequently to the retinal pigment epithelium (RPE) cells. MCMV underwent ocular latency, which was associated with expression of multiple virus genes and from which MCMV could be reactivated by immunosuppression. Latent ocular infection was associated with significant up-regulation of several inflammatory/angiogenic factors. Retinal and choroidal pathologies developed in a progressive manner, with deposits appearing at both basal and apical aspects of the RPE, RPE/choroidal atrophy, photoreceptor degeneration, and neovascularization. The pathologies induced by long-term ocular MCMV latency share features of previously described human ocular diseases, such as age-related macular degeneration.