Mouse Cytomegalovirus Infection Research Articles

Abstract B055: Harnessing cytomegalovirus immunity against pancreatic tumors for immunotherapy

Abstract Immunotherapy has had very limited success in pancreatic cancer, due to its low mutational burden and immunosuppressive microenvironment. Our approach consists in redirecting pre- existing antiviral immunity against pancreatic tumors by delivering viral antigens using the tumor penetrating peptide iRGD .This peptide, targets the tumor vasculature through av integrins and neuropilin-1, delivering conjugated or co-administered cargo to tumors. Here we used mouse cytomegalovirus (CMV) as an infection model. CMV is a β-herpesvirus that induces a strong T-cell response in mice and humans, comprising >10% of all circulating CD4 and CD8 T cells mostly with effector-memory phenotype. Importantly, human CMV infects over 60% of the world’s population rendering it a suitable candidate for translation. Methods: Mice latently infected with CMV were orthotopically implanted with KPC pancreatic tumor cells and treated with systemic injections of vehicle or iRGD plus CMV class I and II binding peptides. Another cohort of age matched uninfected mice was used as a control. Tumor growth and mouse weight were monitored twice a week and CMV specific immune responses were measured in spleen, liver and tumor by flow cytometry, using tetramer staining and CMV specific peptide recall. Results: CMV infected mice receiving iRGD+CMV peptides responded to treatment as evidenced by delayed tumor growth associated with increased tumor necrosis and T cell infiltration. In addition, flow cytometry analysis of tumor, liver and spleen showed a significant increase in CMV specific T cells preferentially in the tumor. Surprisingly, these T cells preferentially infiltrated tumors regardless of the use of iRGD for tumor targeting of CMV peptides. Survival studies showed a marked increase in median survival, reaching 42 days in the infected and treated group, compared to 25 days in the vehicle treated one. The survival benefit was very similar in the presence or absence of iRGD. Combining this strategy with anti- PD1 and or anti-IL10R did not significantly improve the benefits of CMV therapy alone. On the other hand, combination with low dose gemcitabine (5mg/kg) significantly improved tumor growth delay and survival in the combination compared to either therapy alone. The median survival reached 49 days in the combination, compared to 39 for gemcitabine or CMV therapy alone and 32 for vehicle.Conclusions: We have recently performed single cell RNA sequencing in tumors from infected and CMV peptide or vehicle treated mice and will analyze the transcriptional profile and TCR sequence of the tumor infiltrating T cells. Preliminary analysis of this data shows a massive T cell infiltration and an increase in dendritic cells and macrophages in infected and treated mice. Taken together, this data shows we can deliver antigens to pancreatic tumors and elicit an anti-tumor immune response that results in delayed tumor growth and has a survival benefit. This is a mutation agnostic approach with high translational value, since more than 60% of the population has previous CMV immunity. Citation Format: Remi Marrocco, Jay Patel, Rithika Medari, Siming Sun, Kevin Gulay, Alexei Martsinkovskiy, Simon Brunel, Eduardo Lucero-Meza, Evangeline Mose, Andrew Lowy, Chris Benedict, Tatiana Hurtado de Mendoza. Harnessing cytomegalovirus immunity against pancreatic tumors for immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B055.

Long-lived central memory γδ T cells confer protection against murine cytomegalovirus reinfection.

The involvement of γδ TCR-bearing lymphocytes in immunological memory has gained increasing interest due to their functional duality between adaptive and innate immunity. γδ T effector memory (TEM) and central memory (TCM) subsets have been identified, but their respective roles in memory responses are poorly understood. In the present study, we used subsequent mouse cytomegalovirus (MCMV) infections of αβ T cell deficient mice in order to analyze the memory potential of γδ T cells. As for CMV-specific αβ T cells, MCMV induced the accumulation of cytolytic, KLRG1+CX3CR1+ γδ TEM that principally localized in infected organ vasculature. Typifying T cell memory, γδ T cell expansion in organs and blood was higher after secondary viral challenge than after primary infection. Viral control upon MCMV reinfection was prevented when masking γδ T-cell receptor, and was associated with a preferential amplification of private and unfocused TCR δ chain repertoire composed of a combination of clonotypes expanded post-primary infection and, more unexpectedly, of novel expanded clonotypes. Finally, long-term-primed γδ TCM cells, but not γδ TEM cells, protected T cell-deficient hosts against MCMV-induced death upon adoptive transfer, probably through their ability to survive and to generate TEM in the recipient host. This better survival potential of TCM cells was confirmed by a detailed scRNASeq analysis of the two γδ T cell memory subsets which also revealed their similarity to classically adaptive αβ CD8 T cells. Overall, our study uncovered memory properties of long-lived TCM γδ T cells that confer protection in a chronic infection, highlighting the interest of this T cell subset in vaccination approaches.

Distinct developmental pathways generate functionally distinct populations of natural killer cells.

Natural killer (NK) cells function by eliminating virus-infected or tumor cells. Here we identified an NK-lineage-biased progenitor population, referred to as early NK progenitors (ENKPs), which developed into NK cells independently of common precursors for innate lymphoid cells (ILCPs). ENKP-derived NK cells (ENKP_NK cells) and ILCP-derived NK cells (ILCP_NK cells) were transcriptionally different. We devised combinations of surface markers that identified highly enriched ENKP_NK and ILCP_NK cell populations in wild-type mice. Furthermore, Ly49H+ NK cells that responded to mouse cytomegalovirus infection primarily developed from ENKPs, whereas ILCP_NK cells were better IFNγ producers after infection with Salmonella and herpes simplex virus. Human CD56dim and CD56bright NK cells were transcriptionally similar to ENKP_NK cells and ILCP_NK cells, respectively. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets in mice and further suggest these pathways may be conserved in humans.

Virological, innate, and adaptive immune profiles shaped by variation in route and age of host in murine cytomegalovirus infection.

The majority of experiments modeling human cytomegalovirus (hCMV) infection in mice have been carried out using intraperitoneal infection in sexually mature adult mice, which stands in contrast to the large number of humans being infected with human CMV at a young age, most likely via bodily fluids through the nasopharyngeal/oral route. This study examined the impact of the choice of age and route of infection in modeling CMV infection in mice. By comparing young, prepubescent to older sexually mature counterparts, infected either via the intranasal or intraperitoneal route, we discovered substantial differences in deployment and response intensity of different arms of the immune system in systemic control of the virus; tissue responses, by contrast, appeared similar between ages and infection routes.

Abstract 2470: Harnessing cytomegalovirus immunity against pancreatic tumors for immunotherapy

Abstract Immunotherapy has had very limited success in pancreatic cancer, due to its low mutational burden and immunosuppressive microenvironment. Our approach consists in redirecting pre-existing antiviral immunity against pancreatic tumors by delivering viral antigens using the tumor penetrating peptide iRGD .This peptide, targets the tumor vasculature through αv integrins and neuropilin-1, delivering conjugated or co-administered cargo to tumors. Here we used mouse cytomegalovirus (CMV) as an infection model. CMV is a β-herpesvirus that induces a strong T-cell response in mice and humans, comprising >10% of all circulating CD4 and CD8 T cells mostly with effector-memory phenotype. Importantly, human CMV infects over 60% of the world’s population rendering it a suitable candidate for translation. Mice latently infected with CMV were orthotopically implanted with KPC pancreatic tumor cells and treated with systemic injections of vehicle or iRGD plus CMV class I and II binding peptides. Another cohort of age matched uninfected mice was used as a control. Tumor growth and mouse weight were monitored twice a week and CMV specific immune responses were measured in spleen and tumor by flow cytometry, using tetramer staining and CMV specific peptide recall. CMV infected mice receiving iRGD + CMV peptides responded to treatment as evidenced by delayed tumor growth associated with increased tumor necrosis and T cell infiltration. In addition, flow cytometry analysis of tumor and spleen showed a significant increase in CMV specific T cells that produced significant amounts of IFN-γ and TNF-α upon CMV peptide recall. Furthermore, tumor regulatory T cells (Treg) were significantly reduced and the CD8/T reg ratio was greatly increased. Survival studies showed a 68% increase in median survival in the treated and infected group. Combining this strategy with anti-PD1 and or anti-IL10R did not significantly improve the benefits of iRGD + CMV therapy alone. Other combinations with chemotherapy and TME remodeling agents are being tested.Taken together, this data shows we can deliver antigens to pancreatic tumors via iRGD and elicit an anti-tumor immune response that results in delayed tumor growth and has a survival benefit. This is a mutation agnostic approach with high translational value, since more than 60% of the population has previous CMV immunity and iRGD in combination with chemotherapy is already in phase II clinical trials for advanced pancreatic cancer. Citation Format: Remi Marrocco, Jay Patel, Siming Sun, Rithika Medari, Kevin Gulay, Eduardo Lucero-Meza, Evangeline Mose, Andrew Lowy, Chris Benedict, Tatiana Hurtado de Mendoza. Harnessing cytomegalovirus immunity against pancreatic tumors for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2470.

Indirect CD4+ T cell protection against persistent MCMV infection by NK cells requires IFNγ.

Host control of mouse cytomegalovirus (MCMV) infection of MHCII- salivary gland acinar cells is mediated by CD4+ T cells, but how they protect is unclear. Here, we show CD4+ T cells control MCMV indirectly in the salivary gland, via IFNγ engagement with uninfected, but antigen+ MHCII+ APC and recruitment of NK cells to infected cell foci. This immune mechanism renders direct contact of CD4+ T cells with infected cells unnecessary and may represent a host strategy to overcome viral immune evasion.

Late-rising CD4 T cells resolve mouse cytomegalovirus persistent replication in the salivary gland.

Conventional antiviral memory CD4 T cells typically arise during the first two weeks of acute infection. Unlike most viruses, cytomegalovirus (CMV) exhibits an extended persistent replication phase followed by lifelong latency accompanied with some gene expression. We show that during mouse CMV (MCMV) infection, CD4 T cells recognizing an epitope derived from the viral M09 protein only develop after conventional memory T cells have already peaked and contracted. Ablating these CD4 T cells by mutating the M09 genomic epitope in the MCMV Smith strain, or inducing them by introducing the epitope into the K181 strain, resulted in delayed or enhanced control of viral persistence, respectively. These cells were shown to be unique compared to their conventional memory counterparts; producing higher IFNγ and IL-2 and lower IL-10 levels. RNAseq analyses revealed them to express distinct subsets of effector genes as compared to classical CD4 T cells. Additionally, when M09 cells were induced by epitope vaccination they significantly enhanced protection when compared to conventional CD4 T cells alone. These data show that late-rising CD4 T cells are a unique memory subset with excellent protective capacities that display a development program strongly differing from the majority of memory T cells.

Intermittent cytomegalovirus infection alters neurobiological metabolism and induces cognitive deficits in mice

Risk factors contributing to dementia are multifactorial. Accumulating evidence suggests a role for pathogens as risk factors, but data is largely correlative with few causal relationships. Here, we demonstrate that intermittent murine cytomegalovirus (MCMV) infection of mice, alters blood brain barrier (BBB) permeability and metabolic pathways. Increased basal mitochondrial function is observed in brain microvessels cells (BMV) exposed to intermittent MCMV infection and is accompanied by elevated levels of superoxide. Further, mice score lower in cognitive assays compared to age-matched controls who were never administered MCMV. Our data show that repeated systemic infection with MCMV, increases markers of neuroinflammation, alters mitochondrial function, increases markers of oxidative stress and impacts cognition. Together, this suggests that viral burden may be a risk factor for dementia. These observations provide possible mechanistic insights through which pathogens may contribute to the progression or exacerbation of dementia.

Abstract A028: Defining immune pathways involved in the depletion of B-cell precursor acute lymphoblastic leukemia-initiating cells following early-life cytomegalovirus (CMV) infection in mice

Abstract Background: B-cell acute lymphoblastic leukemia (B-ALL) is one of the most common forms of childhood cancers. Epidemiological studies have shown contrasting results regarding the role of infections in the risk of leukemia, and there is still no clear explanation for the pro- or anti-leukemia effects. Aim: This study aimed to investigate the influence of cytomegalovirus (CMV) infection on the development of B-ALL and identify the underlying immune mechanisms involved. Materials and Methods: In this study, Eμ-Ret transgenic mice were used as a model for hyperdiploid B-ALL, and luciferase-tagged CMV was used as a common early-life infection. The impact of infection on different subsets of B cells and leukemic initiating cells (LICs) in the bone marrow and spleen of neonatal and adult mice was assessed using multicolor flow cytometry. To gain insights into the mechanisms that impact LICs after infection, we sorted LICs from the splenocytes of infected and non-infected RFP mice seven days after infection, and total RNA sequencing (RNA-seq) was performed for gene expression analysis. We used neutralizing antibodies to evaluate the role of different cytokines/chemokines in mediating LIC depletion in vivo. Results: Significant infection-induced depletion of preleukemic cells occurred in infected neonate Eμ-ret mice on BALBc, BALBc/FVB (F1) and BALBc/C57 (F1) backgrounds. This depletion showed an age-dependent pattern, with only infection in the first week after birth reducing the LICs numbers. Depletion of preleukemia was dependent on Stat4 and p40 homodimer/monomers, but not p40 heterodimers (IL-12 and IL-23). Several chemokines/cytokines, including IFNg and TNFa, showed high levels of expression in the serum of CMV-infected pups but not in adult mice. RNA sequencing indicated that IFNg and TNFa exerted a direct effect on preleukemic cells. In vivo neutralization of IFNg completely blocked the depletion effect of CMV infection. Further experiments confirmed that this cytokine works downstream of the Stat4 signaling pathway. Conclusion: This is the first model to study the impact of CMV infection and the time of its exposure on B-ALL initiating cells. The identified Stat4-, IL12p40 homodimer/monomer-, and IFNg- driven activities could inform future treatment and prevention approaches for B-ALL. Citation Format: Ali Farrokhi, Tanmaya Atre, Citlali Marquez1, Soren Gantt, Gregor Reid. Defining immune pathways involved in the depletion of B-cell precursor acute lymphoblastic leukemia-initiating cells following early-life cytomegalovirus (CMV) infection in mice [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A028.

Themis2 regulates natural killer cell memory function and formation

Immunological memory is a hallmark of the adaptive immune system. Although natural killer (NK) cells are innate immune cells important for the immediate host defence, they can differentiate into memory NK cells. The molecular mechanisms controlling this differentiation are yet to be fully elucidated. Here we identify the scaffold protein Themis2 as a critical regulator of memory NK cell differentiation and function. Themis2-deficient NK cells expressing Ly49H, an activating NK receptor for the mouse cytomegalovirus (MCMV) antigen m157, show enhanced differentiation into memory NK cells and augment host protection against MCMV infection. Themis2 inhibits the effector function of NK cells after stimulation of Ly49H and multiple activating NK receptors, though not specific to memory NK cells. Mechanistically, Themis2 suppresses Ly49H signalling by attenuating ZAP70/Syk phosphorylation, and it also translocates to the nucleus, where it promotes Zfp740-mediated repression to regulate the persistence of memory NK cells. Zfp740 deficiency increases the number of memory NK cells and enhances the effector function of memory NK cells, which further supports the relevance of the Themis2-Zfp740 pathway. In conclusion, our study shows that Themis2 quantitatively and qualitatively regulates NK cell memory formation.

Cutting Edge: STAT4 Promotes Bhlhe40 Induction to Drive Protective IFN-γ from NK Cells during Viral Infection.

NK cells represent a cellular component of the mammalian innate immune system, and they mount rapid responses against viral infection, including the secretion of the potent antiviral effector cytokine IFN-γ. Following mouse CMV infection, Bhlhe40 was the most highly induced transcription factor in NK cells among the basic helix-loop-helix family. Bhlhe40 upregulation in NK cells depended upon IL-12 and IL-18 signals, with the promoter of Bhlhe40 enriched for STAT4 and the permissive histone H3K4me3, and with STAT4-deficient NK cells showing an impairment of Bhlhe40 induction and diminished H3K4me3. Transcriptomic and protein analysis of Bhlhe40-deficient NK cells revealed a defect in IFN-γ production during mouse CMV infection, resulting in diminished protective immunity following viral challenge. Finally, we provide evidence that Bhlhe40 directly promotes IFN-γ by binding throughout the Ifng loci in activated NK cells. Thus, our study reveals how STAT4-mediated control of Bhlhe40 drives protective IFN-γ secretion by NK cells during viral infection.

Perinatal murine cytomegalovirus infection reshapes the transcriptional profile and functionality of NK cells

Infections in early life can elicit substantially different immune responses and pathogenesis than infections in adulthood. Here, we investigate the consequences of murine cytomegalovirus infection in newborn mice on NK cells. We show that infection severely compromised NK cell maturation and functionality in newborns. This effect was not due to compromised virus control. Inflammatory responses to infection dysregulated the expression of major transcription factors governing NK cell fate, such as Eomes, resulting in impaired NK cell function. Most prominently, NK cells from perinatally infected mice have a diminished ability to produce IFN-γ due to the downregulation of long non-coding RNA Ifng-as1 expression. Moreover, the bone marrow’s capacity to efficiently generate new NK cells is reduced, explaining the prolonged negative effects of perinatal infection on NK cells. This study demonstrates that viral infections in early life can profoundly impact NK cell biology, including long-lasting impairment in NK cell functionality.

Control of nutrient uptake by IRF4 orchestrates innate immune memory.

Natural killer (NK) cells are innate cytotoxic lymphocytes with adaptive immune features, including antigen specificity, clonal expansion and memory. As such, NK cells share many transcriptional and epigenetic programs with their adaptive CD8+ T cell siblings. Various signals ranging from antigen, co-stimulation and proinflammatory cytokines are required for optimal NK cell responses in mice and humans during virus infection; however, the integration of these signals remains unclear. In this study, we identified that the transcription factor IRF4 integrates signals to coordinate the NK cell response during mouse cytomegalovirus infection. Loss of IRF4 was detrimental to the expansion and differentiation of virus-specific NK cells. This defect was partially attributed to the inability of IRF4-deficient NK cells to uptake nutrients required for survival and memory generation. Altogether, these data suggest that IRF4 is a signal integrator that acts as a secondary metabolic checkpoint to orchestrate the adaptive response of NK cells during viral infection.

Combined Host-Pathogen Fate Mapping to Investigate Lung Macrophages in Viral Infection.

Macrophage identity, as defined by epigenetic, transcriptional, proteomic, and functional programs, is greatly impacted by cues originating from the microenvironment. As a consequence, immunophenotyping based on surface marker expression is established and reliable in homeostatic conditions, whereas environmental challenges, in particular infections, severely hamper the determination of identity states. This has become more evident with recent discoveries that macrophage-inherent plasticity may go beyond limits of lineage-defining immunophenotypes. Therefore, transgenic fate mapping tools, such as the phage-derived loxP-cre-system, are essential forthe analysis of macrophage adaptation in the tissue under extreme environmental conditions, for example, upon encounter with pathogens. In this chapter, we describe an advanced application of the loxP-cre-system during infection. Here, the host encodes a cell type-specific cre-recombinase, while the pathogen harbors a STOP-floxed fluorescent reporter gene. As an instructive example for the versatility of the system, we demonstrate that alveolar macrophages are predominantly targeted after respiratory tract infection with mouse cytomegalovirus (MCMV). Combined host-pathogen fate mapping not only enables to distinguish between infected and non-infected (bystander) macrophages but also spurs exploration of phenotypic adaptation and tracing of cellular localization in the context of MCMV infection. Moreover, we provide a gating strategy for resolving the diversity of pulmonary immune cell populations.

NK Cells from Human Cytomegalovirus-Seropositive Individuals Have a Distinct Metabolic Profile That Correlates with Elevated mTOR Signaling.

CMV can elicit adaptive immune features in both mouse and human NK cells. Mouse Ly49H+ NK cells expand 100- to 1000-fold in response to mouse CMV infection and persist for months after exposure. Human NKG2C+ NK cells also expand after human CMV (HCMV) infection and persist for months. The clonal expansion of adaptive NK cells is likely an energy-intensive process, and the metabolic requirements that support adaptive NK cell expansion and persistence remain largely uncharacterized. We previously reported that NK cells from HCMV-seropositive donors had increased maximum capacity for both glycolysis and mitochondrial oxidative phosphorylation relative to NK cells from HCMV-seronegative donors. In this article, we report an extension of this work in which we analyzed the metabolomes of NK cells from HCMV-seropositive donors with NKG2C+ expansions and NK cells from HCMV seronegative donors without such expansions. NK cells from HCMV+ donors exhibited striking elevations in purine and pyrimidine deoxyribonucleotides, along with moderate increases in plasma membrane components. Mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that, as a part of mTOR complex 1 (mTORC1), bridges nutrient signaling to metabolic processes necessary for cell growth. Signaling through mTORC1 induces both nucleotide and lipid synthesis. We observed elevated mTORC1 signaling on activation in both NKG2C- and NKG2C+ NK cells from HCMV+ donors relative to those from HCMV- donors, demonstrating a correlation between higher mTORC1 activity and synthesis of key metabolites for cell growth and division.

STAT3 operates as a rheostat regulating sensitivity of NK cells to inflammatory cytokines

Abstract NK cells are innate lymphocytes that respond to a variety of cytokines during viral infection. Several homeostatic and inflammatory cytokines bind to receptors that signal via STAT3. STAT3 can form homodimers or heterodimers. To understand the role of STAT3 homodimers and heterodimers, we made use of knockout models in which NK cells lack STAT3 or specific cytokine receptors. We found that NK cells deficient in STAT3 expand less than wildtype NK cells during mouse cytomegalovirus (MCMV) infection. IL-21R and IL-10R are the only receptors expressed on NK cells that signal through STAT3 homodimers. However, IL-21R or IL-10R-deficient NK cells showed no or little expansion differences, suggesting additional cytokine receptors may be signaling through STAT3. To evaluate whether STAT3 regulates the sensitivity of NK cells to cytokines that signal through heterodimerization partners STAT1 and STAT5, we performed cytokine titrations. We found that sensitivity towards IFN-a and IL-15 was reduced in STAT3-deficient NK cells. NK cells require inflammatory cytokines for antiviral expansion but proliferate poorly in hyper-inflammatory environments. Because STAT3 appears to regulate the sensitivity of NK cells to inflammatory cytokines, we hypothesized that STAT3-deficient NK cells would be shielded in settings of hyper-inflammation. Indeed, using high-dose MCMV infection, we observed that STAT3-deficient NK cells expanded better than WT NK cells. RNA-seq analysis further corroborated that in high-dose infection STAT3-deficient NK cells are nearly indistinguishable from WT NK cells during regular-dose infection. Thus, we believe STAT3 operates as a rheostat, modulating the sensitivity of NK cells to their inflammatory environment. Supported by the Cancer Research Institute, Donald J. Gogel Postdoctoral Fellowship, CRI393

MEF2C is a critical regulator of human NK cell metabolism.

Abstract Natural killer (NK) cells are critical for the first line of defense against viral infection and malignancy. While the transcriptional and epigenetic regulation of activated NK cells is well characterized in mice, these processes are not yet fully understood in human NK cells. We performed a targeted CRISPR/Cas9 ribonucleoprotein (RNP) transcription factor screen in mature primary human NK cells to identify putative transcription factors required for optimal effector function. The transcription factor myocyte enhancer factor 2C (MEF2C) emerged as a previously unidentified regulator of NK cell homeostasis, cytokine production, and cytotoxicity. MEF2C-deficient NK cells activated with IL-2 and IL-15 displayed impaired proliferation, degranulation, and production of granzyme B. CRISPR-mediated deletion of MEF2C resulted in disrupted glycolysis and oxidative phosphorylation using both SCENITH and Seahorse extracellular flux analysis. Liquid chromatography/mass spectrometry (LC/MS)-based metabolomics of MEF2C-deficient NK cells revealed reductions in the metabolites glyceraldehyde-3-phosphate and a-ketoglutarate/succinate, accompanied by increased accumulation of lipid metabolism products including phosphorylethanolamine and saturated long-chain fatty acids. In vivo, CRISPR/Cas9 RNP-mediated disruption of MEF2C expression in mouse NK cells resulted in decreased expansion during mouse cytomegalovirus infection. Together, these studies reveal MEF2C as a novel regulator of NK cell effector function through control of multiple critical cell-intrinsic metabolic pathways.

Murine Cytomegalovirus Infection Induced miR-1929-3p Down-Regulation Promotes the Proliferation and Apoptosis of Vascular Smooth Muscle Cells in Mice by Targeting Endothelin A Receptor and Downstream NLRP3 Activation Pathway.

Our previous study demonstrated in vivo that mouse cytomegalovirus (MCMV) infection promoted vascular remodeling after downregulation of miR-1929-3p. This study aimed to investigate the role of miR-1929-3p/ETAR/NLRP3 pathway in mouse vascular smooth muscle cells (MOVAS) after MCMV infection. First, PCR was used to detect the success of the infection. Second, MOVAS were transfected with the miR-1929-3p mimic, inhibitor, and ETAR overexpressed adenovirus vector. Cell proliferation was detected using EdU, whereas apoptosis was detected using flow cytometry. The expression of miR-1929-3p and ETAR were detected using qRT-PCR. Western blot detected proteins of cell proliferation, apoptosis, and the NLRP3 inflammasome. Interleukin-1β and interleukin-18 were determined using ELISA. The results revealed that after 48h, MCMV infection promoted the proliferation of MOVAS when the MOI was 0.01. MCMV infection increased ETAR by downregulating miR-1929-3p. The miR-1929-3p mimic reversed the proliferation and apoptosis, whereas the miR-1929-3p inhibitor promoted this effect. ETAR overexpression further promoted MCMV infection by downregulating miR-1929-3p-mediated proliferation and apoptosis. MCMV infection mediates the downregulation of miR-1929-3p and the upregulation of ETAR, which activates NLRP3 inflammasome. In conclusion, MCMV infection promoted the proliferation of MOVAS, possibly by downregulating miR-1929-3p, promoting the upregulation of the target gene ETAR and activating NLRP3 inflammasome.

Research progress of deafness induced by cytomegalovirus infection in mice

巨细胞病毒（cytomegalovirus，CMV）是最常见的先天性感染病原体之一，先天性CMV感染最易引起感音神经性听力损失（sensorineural hearing loss，SNHL）。本文重点围绕鼠CMV（murine CMV，MCMV）感染小鼠致聋模型构建、CMV感染致聋机制以及靶点药物的筛选进行综述，认为：（1）小鼠模型可作为研究CMV感染致聋机制较好的工具；（2）CMV感染的致聋机制主要涉及内耳细胞损伤、细胞丢失、细胞凋亡、炎症反应、钙信号系统异常和宿主免疫反应等；（3）更昔洛韦、双黄连和黄连素可改善MCMV诱导的听力损失，以期为临床预防和治疗CMV感染引起的SNHL提供参考。.

NPP-669, a Novel Broad-Spectrum Antiviral Therapeutic with Excellent Cellular Uptake, Antiviral Potency, Oral Bioavailability, Preclinical Efficacy, and a Promising Safety Margin.

DNA viruses are responsible for many diseases in humans. Current treatments are often limited by toxicity, as in the case of cidofovir (CDV, Vistide), a compound used against cytomegalovirus (CMV) and adenovirus (AdV) infections. CDV is a polar molecule with poor bioavailability, and its overall clinical utility is limited by the high occurrence of acute nephrotoxicity. To circumvent these disadvantages, we designed nine CDV prodrug analogues. The prodrugs modulate the polarity of CDV with a long sulfonyl alkyl chain attached to one of the phosphono oxygens. We added capping groups to the end of the alkyl chain to minimize β-oxidation and focus the metabolism on the phosphoester hydrolysis, thereby tuning the rate of this reaction by altering the alkyl chain length. With these modifications, the prodrugs have excellent aqueous solubility, optimized metabolic stability, increased cellular permeability, and rapid intracellular conversion to the pharmacologically active diphosphate form (CDV-PP). The prodrugs exhibited significantly enhanced antiviral potency against a wide range of DNA viruses in infected human foreskin fibroblasts. Single-dose intravenous and oral pharmacokinetic experiments showed that the compounds maintained plasma and target tissue levels of CDV well above the EC50 for 24 h. These experiments identified a novel lead candidate, NPP-669. NPP-669 demonstrated efficacy against CMV infections in mice and AdV infections in hamsters following oral (p.o.) dosing at a dose of 1 mg/kg BID and 0.1 mg/kg QD, respectively. We further showed that NPP-669 at 30 mg/kg QD did not exhibit histological signs of toxicity in mice or hamsters. These data suggest that NPP-669 is a promising lead candidate for a broad-spectrum antiviral compound.