The Cytomegalovirus M35 Protein Directly Binds to the Interferon-ß Enhancer and Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes.

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerization is a key feature for M35's immunomodulatory activity. In electrophoretic mobility shift assays (EMSAs), purified M35 protein specifically bound to the regulatory DNA element that governs transcription of the first type I IFN gene induced in nonimmune cells, Ifnb1. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signaling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signaling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labeled transcripts (SLAM-seq) and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically downregulated basal expression of IRF3-dependent genes. During MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonizes gene induction mediated by IRF3 and impairs the antiviral response more broadly than formerly recognized. IMPORTANCE Replication of the ubiquitous human cytomegalovirus (HCMV) in healthy individuals mostly goes unnoticed but can impair fetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry into host cells, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here, we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key cellular factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.

Exogenous Interleukin-37 Alleviates Hepatitis with Reduced Dendritic Cells and Induced Regulatory T Cells in Acute Murine Cytomegalovirus Infection.

Human cytomegalovirus (HCMV) infection is globally distributed, and the liver is one of the major targeting organs. So far, the mechanisms for cell and organ damage have not fully been elucidated and the treatments for the infection are mainly at symptoms. IL-37 has shown a protective role in certain inflammatory diseases. In the present study, potential protective effect of exogenous IL-37 on murine cytomegalovirus- (MCMV-) infected hepatitis was evaluated through analyses of serum transaminases, the liver histopathology and cytokine expression, and functional state of dendritic cells (DCs) and regulatory T cells (Tregs). These analyses showed a significant decrease in serum transaminase levels and a lower Ishak histopathologic score at the early stage of MCMV-infected mice with exogenous IL-37 pretreatment. The frequencies of MHC-Ⅱ, CD40, CD80, and CD86 positive DCs in the liver and spleen were decreased significantly at 7 days postinfection (dpi) in MCMV-infected mice with IL-37 pretreatment when compared with those without the pretreatment, while the total number of DCs in the liver was reduced in IL-37-pretreated mice. The induction of Tregs in the spleen was enhanced at dpi 3 with IL-37 pretreatment in MCMV-infected mice. The mRNA expression levels of cytokines in the liver were decreased significantly (IL-1ß, IL-6, IL-10, IL-4) or to some extent (TGF-ß and TNF-α). The present study suggested that exogenous IL-37 can alleviate MCMV-infected hepatitis, likely through reduced DCs and induced Tregs with a weaker cytokine storm, demonstrating its potential value in clinical management for HCMV-infected hepatitis.

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-1ß and interleukin-18 were determined using ELISA. The results revealed that after 48 h, 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.

Membraneless Compartmentalization of Nuclear Assembly Sites during Murine Cytomegalovirus Infection.

Extensive reorganization of infected cells and the formation of large structures known as the nuclear replication compartment (RC) and cytoplasmic assembly compartment (AC) is a hallmark of beta-herpesvirus infection. These restructurings rely on extensive compartmentalization of the processes that make up the virus manufacturing chain. Compartmentalization of the nuclear processes during murine cytomegalovirus (MCMV) infection is not well described. In this study, we visualized five viral proteins (pIE1, pE1, pM25, pm48.2, and pM57) and replicated viral DNA to reveal the nuclear events during MCMV infection. As expected, these events can be matched with those described for other beta and alpha herpesviruses and contribute to the overall picture of herpesvirus assembly. Imaging showed that four viral proteins (pE1, pM25, pm48.2, and pM57) and replicated viral DNA condense in the nucleus into membraneless assemblies (MLAs) that undergo a maturation sequence to form the RC. One of these proteins (pM25), which is also expressed in a cytoplasmic form (pM25l), showed similar MLAs in the AC. Bioinformatics tools for predicting biomolecular condensates showed that four of the five proteins had a high propensity for liquid-liquid phase separation (LLPS), suggesting that LLPS may be a mechanism for compartmentalization within RC and AC. Examination of the physical properties of MLAs formed during the early phase of infection by 1,6-hexanediol treatment in vivo revealed liquid-like properties of pE1 MLAs and more solid-like properties of pM25 MLAs, indicating heterogeneity of mechanisms in the formation of virus-induced MLAs. Analysis of the five viral proteins and replicated viral DNA shows that the maturation sequence of RC and AC is not completed in many cells, suggesting that virus production and release is carried out by a rather limited number of cells. This study thus lays the groundwork for further investigation of the replication cycle of beta-herpesviruses, and the results should be incorporated into plans for high-throughput and single-cell analytic approaches.

Low expression of miR-1929-3p mediates murine cytomegalovirus-induced fibrosis in cardiac fibroblasts via targeting endothelin a receptor/NLRP3 inflammasome pathway.

MicroRNAs are crucial in the development of myocardial remodeling in hypertension. Low miR-1929-3p expression induced by murine cytomegalovirus (MCMV) infection is closely related to hypertensive myocardial remodeling. This study investigated the molecular mechanism of miR-1929-3p-induced myocardial remodeling after MCMV infection. We modeled MCMV-infected mouse cardiac fibroblasts (MMCFs) as the primary cell model. First, MCMV infection reduced the expression of miR-1929-3p and increased the mRNA and protein expression of its target gene endothelin receptor type A (ETAR) in mouse cardiac fibroblasts (MCFs), which demonstrated an internal relationship with myocardial fibrosis (MF) based on high proliferation, phenotypic transformation (α-SMA), and collagen expression in MMCFs. The transfection of the miR-1929-3p mimic downregulated the high expression of ETAR and alleviated these adverse effects in MMCFs. Inversely, these effects were exacerbated by the miR-1929-3p inhibitor. Second, the transfection of endothelin receptor type A over-expressed adenovirus (adETAR) reversed these positive effects of the miR-1929-3p mimic on MF improvement. Third, the transfection of adETAR exhibited a strong inflammatory response in MMCFs with increased expression of NOD-like receptors pyrin domain containing 3 (NLRP3) and increased secretion of interleukin-18. However, we found that the ETAR antagonist BQ123 and the selected NLRP3 inflammasome inhibitor MCC950 effectively eliminated the inflammatory response induced by both MCMV infection and miR-1929-3p inhibitor. Moreover, the MCF supernatant was related to cardiomyocyte hypertrophy. Our findings suggest that MCMV infection promotes MF by inducing the downregulation of miR-1929-3p and the high expression of ETAR, which activates NLRP3 inflammasomes in MCFs.

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.

Tegument Protein pp150 Sequence-Specific Peptide Blocks Cytomegalovirus Infection.

Human cytomegalovirus (HCMV) tegument protein pp150 is essential for the completion of the final steps in virion maturation. Earlier studies indicated that three pp150nt (N-terminal one-third of pp150) conformers cluster on each triplex (Tri1, Tri2A and Tri2B), and extend towards small capsid proteins atop nearby major capsid proteins, forming a net-like layer of tegument densities that enmesh and stabilize HCMV capsids. Based on this atomic detail, we designed several peptides targeting pp150nt. Our data show significant reduction in virus growth upon treatment with one of these peptides (pep-CR2) with an IC50 of 1.33 µM and no significant impact on cell viability. Based on 3D modeling, pep-CR2 specifically interferes with the pp150-capsid binding interface. Cells pre-treated with pep-CR2 and infected with HCMV sequester pp150 in the nucleus, indicating a mechanistic disruption of pp150 loading onto capsids and subsequent nuclear egress. Furthermore, pep-CR2 effectively inhibits mouse cytomegalovirus (MCMV) infection in cell culture, paving the way for future animal testing. Combined, these results indicate that CR2 of pp150 is amenable to targeting by a peptide inhibitor, and can be developed into an effective antiviral.

MicroRNA­1929­3p participates in murine cytomegalovirus­induced hypertensive vascular remodeling through Ednra/NLRP3 inflammasome activation.

MicroRNAs (miRNAs) play an important role in the development of vascular remodeling in essential hypertension (EH) by mediating the effects of human cytomegalovirus (HCMV) on the vascular system. Therefore, the aim of the present study was to investigate the effects of murine cytomegalovirus (MCMV) infection on blood pressure and vascular function in mice, in order to elucidate the role of miR­1929­3p in this process. For model development, 7­month­old C57BL/6J mice were infected with the Smith strain of MCMV, and MCMV DNA, IgG and IgM were detected. Subsequently, blood pressure was measured via the carotid artery, and the morphological changes of the aorta were evaluated by hematoxylin and eosin and Masson's trichrome staining. miR­1929­3p transfection was performed using an adeno­associated virus packaged vector and the changes in vascular structure were then observed. The levels of nitric oxide (NO) and endothelial NO synthase were also assessed with colorimetry. Vascular remodeling and expression of NLRP3 inflammasome pathway­related proteins were detected by immunohistochemistry and western blotting. Endothelin­1 (ET­1), interleukin (IL)­1ß and IL­18 were assayed by ELISA. The results revealed that MCMV infection increased the blood pressure, promoted vascular remodeling, caused endothelial cell injury, and downregulated miR­1929­3p. However, these effects were alleviated by miR­1929­3p overexpression, which downregulated endothelin A receptor (Ednra) and NLRP3 inflammasome, as well as endothelial injury­ and vascular remodeling­related genes. Taken together, the findings of the present study indicated that overexpression of miR­1929­3p may improve MCMV­induced vascular remodeling, possibly via the deactivation of the NLRP3 inflammasome by ET­1/Ednra.

Necroptosis-based CRISPR knockout screen reveals Neuropilin-1 as a critical host factor for early stages of murine cytomegalovirus infection.

Herpesviruses are ubiquitous human pathogens that cause a wide range of health complications. Currently, there is an incomplete understanding of cellular factors that contribute to herpesvirus infection. Here, we report an antiviral necroptosis-based genetic screen to identify novel host cell factors required for infection with the ß-herpesvirus murine cytomegalovirus (MCMV). Our genome-wide CRISPR-based screen harnessed the capacity of herpesvirus mutants that trigger antiviral necroptotic cell death upon early viral gene expression. Vascular endothelial growth factor (VEGF) and semaphorin-binding receptor Neuropilin-1 (Nrp-1) emerge as crucial determinants of MCMV infection. We find that elimination of Nrp-1 impairs early viral gene expression and reduces infection rates in endothelial cells, fibroblasts, and macrophages. Furthermore, preincubation of virus with soluble Nrp-1 dramatically inhibits infection by reducing virus attachment. Thus, Nrp-1 is a key determinant of the initial phase of MCMV infection.

Tetramer Immunization and Selection Followed by CELLISA Screening to Generate Monoclonal Antibodies against the Mouse Cytomegalovirus m12 Immunoevasin.

The generation of reliable mAb of unique and desired specificities serves as a valuable technology to study protein expression and function. However, standard approaches to mAb generation usually involve large-scale protein purification and intensive screening. In this study, we describe an optimized high-throughput proof-of-principle method for the expanded generation, enrichment, and screening of mouse hybridomas secreting mAb specific for a protein of interest. Briefly, we demonstrate that small amounts of a biotinylated protein of interest can be used to generate tetramers for use as prime-boost immunogens, followed by selective enrichment of Ag-specific B cells by magnetic sorting using the same tetramers prior to hybridoma generation. This serves two purposes: 1) to effectively expand both low- and high-affinity B cells specific for the antigenic bait during immunization and 2) to minimize subsequent laborious hybridoma efforts by positive selection of Ag-specific, Ab-secreting cells prior to hybridoma fusion and validation screening. Finally, we employ a rapid and inexpensive screening technology, CELLISA, a high-throughput validation method that uses a chimeric Ag fused to the CD3ζ signaling domain expressed on enzyme-generating reporter cells; these reporters can detect specific mAb in hybridoma supernatants via plate-bound Ab-capture arrays, thereby easing screening. Using this strategy, we generated and characterized novel mouse mAb specific for a viral immunoevasin, the mouse CMV m12 protein, and suggest that these mAb may protect mice from CMV infection via passive immunity.

Murine Cytomegalovirus M25 Proteins Sequester the Tumor Suppressor Protein p53 in Nuclear Accumulations.

To ensure productive infection, herpesviruses utilize tegument proteins and nonstructural regulatory proteins to counteract cellular defense mechanisms and to reprogram cellular pathways. The M25 proteins of mouse cytomegalovirus (MCMV) belong to the betaherpesvirus UL25 gene family that encodes viral proteins implicated with regulatory functions. Through affinity purification and mass spectrometric analysis, we discovered the tumor suppressor protein p53 as a host factor interacting with the M25 proteins. M25-p53 interaction in infected and transfected cells was confirmed by coimmunoprecipitation. Moreover, the proteins colocalized in nuclear dot-like structures upon both infection and inducible expression of the two M25 isoforms. p53 accumulated in wild-type MCMV-infected cells, while this did not occur upon infection with a mutant lacking the M25 gene. Both M25 proteins were able to mediate the effect, identifying them as the first CMV proteins responsible for p53 accumulation during infection. Interaction with M25 proteins led to substantial prolongation of the half-life of p53. In contrast to the higher abundance of the p53 protein in wild-type MCMV-infected cells, the transcript levels of the prominent p53 target genes Cdkn1a and Mdm2 were diminished compared to cells infected with the ΔM25 mutant, and this was associated with reduced binding of p53 to responsive elements within the respective promoters. Notably, the productivity of the M25 deletion mutant was partially rescued on p53-negative fibroblasts. We propose that the MCMV M25 proteins sequester p53 molecules in the nucleus of infected cells, reducing their availability for activating a subset of p53-regulated genes, thereby dampening the antiviral role of p53.IMPORTANCE Host cells use a number of factors to defend against viral infection. Viruses are, however, in an arms race with their host cells to overcome these defense mechanisms. The tumor suppressor protein p53 is an important sensor of cell stress induced by oncogenic insults or viral infections, which upon activation induces various pathways to ensure the integrity of cells. Viruses have to counteract many functions of p53, but complex DNA viruses such as cytomegaloviruses may also utilize some p53 functions for their own benefit. In this study, we discovered that the M25 proteins of mouse cytomegalovirus interact with p53 and mediate its accumulation during infection. Interaction with the M25 proteins sequesters p53 molecules in nuclear dot-like structures, limiting their availability for activation of a subset of p53-regulated target genes. Understanding the interaction between viral proteins and p53 may allow to develop new therapeutic strategies against cytomegalovirus and other viruses.

Cytomegalovirus inhibition of extrinsic apoptosis determines fitness and resistance to cytotoxic CD8 T cells.

Viral immune evasion is currently understood to focus on deflecting CD8 T cell recognition of infected cells by disrupting antigen presentation pathways. We evaluated viral interference with the ultimate step in cytotoxic T cell function, the death of infected cells. The viral inhibitor of caspase-8 activation (vICA) conserved in human cytomegalovirus (HCMV) and murine CMV (MCMV) prevents the activation of caspase-8 and proapoptotic signaling. We demonstrate the key role of vICA from either virus, in deflecting antigen-specific CD8 T cell-killing of infected cells. vICA-deficient mutants, lacking either UL36 or M36, exhibit greater susceptibility to CD8 T cell control than mutants lacking the set of immunoevasins known to disrupt antigen presentation via MHC class I. This difference is evident during infection in the natural mouse host infected with MCMV, in settings where virus-specific CD8 T cells are adoptively transferred. Finally, we identify the molecular mechanism through which vICA acts, demonstrating the central contribution of caspase-8 signaling at a point of convergence of death receptor-induced apoptosis and perforin/granzyme-dependent cytotoxicity.

Murine Cytomegalovirus Protein pM49 Interacts with pM95 and Is Critical for Viral Late Gene Expression.

Late gene expression of betaherpesviruses and gammaherpesviruses is tightly controlled by virus-encoded transactivation factors (vTFs). We recently proved that the 6 vTFs of murine cytomegalovirus (MCMV) form a complex to regulate late gene transcription. pM49, one of the vTFs that has not been studied before, was identified to be a component of the complex that interacts with pM95. In this study, we began to investigate the potential role of pM49 in viral late gene expression. A recombinant MCMV expressing C-terminal FLAG-tagged pM49 was constructed to study the expression kinetics and localization of pM49. pM49 was expressed at the late time of virus infection. Inhibition of viral DNA synthesis by phosphonate sodium phosphonic acid (PAA) abolished pM49 expression, indicating that it is a late protein. pM49 colocalized with pM44 at the viral replication compartment, similarly to other viral vTFs that have been reported. Mutant virus lacking full-length pM49 expression failed to express viral late genes, leading to nonproductive infection. The expression of immediate early and early genes was not affected, and viral DNA synthesis was only minimally affected during pM49-deficient virus infection. All of these data support the role of pM49 in viral late gene expression. After a series of mutagenesis analyses, two key residues, K325 and C326, were identified as required for pM49-pM95 interaction. Cells expressing pM49 with either single mutation of these two residues failed to rescue the late gene expression and support the replication of pM49-deficient virus. Our results indicated that pM49-pM95 interaction is essential for viral late gene expression.IMPORTANCE Cytomegalovirus (CMV) infections result in morbidity and mortality in immunocompromised individuals, and the virus is also a major cause of birth defects in newborns. Currently, because of the unavailability of vaccines against this virus and restricted antiviral therapies with low toxicity, as well as the emergency of resistant strain of this virus, the understanding of viral late gene regulation may provide clues to study new antiviral drugs or vaccines. In this study, we report that MCMV protein pM49 is critical for viral late gene transcription, based on its interaction with pM95. This finding reveals the important role of pM49-pM95 interaction in the regulation of viral late gene expression and that it could be a future potential target for therapeutic intervention in CMV diseases.

Herpesviruses induce aggregation and selective autophagy of host signalling proteins NEMO and RIPK1 as an immune-evasion mechanism.

Viruses manipulate cellular signalling by inducing the degradation of crucial signal transducers, usually via the ubiquitin-proteasome pathway. Here, we show that the murine cytomegalovirus (Murid herpesvirus 1) M45 protein induces the degradation of two cellular signalling proteins, the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) essential modulator (NEMO) and the receptor-interacting protein kinase 1 (RIPK1), via a different mechanism: it induces their sequestration as insoluble protein aggregates and subsequently facilitates their degradation by autophagy. Aggregation of target proteins requires a distinct sequence motif in M45, which we termed 'induced protein aggregation motif'. In a second step, M45 recruits the retromer component vacuolar protein sorting 26B (VPS26B) and the microtubule-associated protein light chain 3 (LC3)-interacting adaptor protein TBC1D5 to facilitate degradation of aggregates by selective autophagy. The induced protein aggregation motif is conserved in M45-homologous proteins of several human herpesviruses, including herpes simplex virus, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, but is only partially conserved in the human cytomegalovirus UL45 protein. We further show that the HSV-1 ICP6 protein induces RIPK1 aggregation and degradation in a similar fashion to M45. These data suggest that induced protein aggregation combined with selective autophagy of aggregates (aggrephagy) represents a conserved viral immune-evasion mechanism.

The complex of MCMV proteins and MHC class I evades NK cell control and drives the evolution of virus-specific activating Ly49 receptors.

CMVs efficiently target MHC I molecules to avoid recognition by cytotoxic T cells. However, the lack of MHC I on the cell surface renders the infected cell susceptible to NK cell killing upon missing self recognition. To counter this, mouse CMV (MCMV) rescues some MHC I molecules to engage inhibitory Ly49 receptors. Here we identify a new viral protein, MATp1, that is essential for MHC I surface rescue. Rescued altered-self MHC I molecules show increased affinity to inhibitory Ly49 receptors, resulting in inhibition of NK cells despite substantially reduced MHC I surface levels. This enables the virus to evade recognition by licensed NK cells. During evolution, this novel viral immune evasion mechanism could have prompted the development of activating NK cell receptors that are specific for MATp1-modified altered-self MHC I molecules. Our study solves a long-standing conundrum of how MCMV avoids recognition by NK cells, unravels a fundamental new viral immune evasion mechanism, and demonstrates how this forced the evolution of virus-specific activating MHC I-restricted Ly49 receptors.

Immunometabolic phenotype of BV-2 microglia cells upon murine cytomegalovirus infection.

Microglia are resident brain macrophages with key roles in development and brain homeostasis. Cytomegalovirus (CMV) readily infects microglia cells, even as a possible primary target of infection in development. Effects of CMV infection on a cellular level in microglia are still unclear; therefore, the aim of this research was to assess the immunometabolic changes of BV-2 microglia cells following the murine cytomegalovirus (MCMV) infection. In light of that aim, we established an in vitro model of ramified BV-2 microglia (BV-2∅FCS, inducible nitric oxide synthase (iNOSlow), arginase-1 (Arg-1high), mannose receptor CD206high, and hypoxia-inducible factor 1α (HIF-1αlow)) to better replicate the in vivo conditions by removing FCS from the cultivation media, while the cells cultivated in 10% FCS DMEM displayed an ameboid morphology (BV-2FCS high, iNOShigh, Arg-1low, CD206low, and HIF-1αhigh). Experiments were performed using both ramified and ameboid microglia, and both of them were permissive to productive viral infection. Our results indicate that MCMV significantly alters the immunometabolic phenotypic properties of BV-2 microglia cells through the manipulation of iNOS and Arg-1 expression patterns, along with an induction of a glycolytic shift in the infected cell cultures.

Atomic structures and deletion mutant reveal different capsid-binding patterns and functional significance of tegument protein pp150 in murine and human cytomegaloviruses with implications for therapeutic development.

Cytomegalovirus (CMV) infection causes birth defects and life-threatening complications in immunosuppressed patients. Lack of vaccine and need for more effective drugs have driven widespread ongoing therapeutic development efforts against human CMV (HCMV), mostly using murine CMV (MCMV) as the model system for preclinical animal tests. The recent publication (Yu et al., 2017, DOI: 10.1126/science.aam6892) of an atomic model for HCMV capsid with associated tegument protein pp150 has infused impetus for rational design of novel vaccines and drugs, but the absence of high-resolution structural data on MCMV remains a significant knowledge gap in such development efforts. Here, by cryoEM with sub-particle reconstruction method, we have obtained the first atomic structure of MCMV capsid with associated pp150. Surprisingly, the capsid-binding patterns of pp150 differ between HCMV and MCMV despite their highly similar capsid structures. In MCMV, pp150 is absent on triplex Tc and exists as a "Λ"-shaped dimer on other triplexes, leading to only 260 groups of two pp150 subunits per capsid in contrast to 320 groups of three pp150 subunits each in a "Δ"-shaped fortifying configuration. Many more amino acids contribute to pp150-pp150 interactions in MCMV than in HCMV, making MCMV pp150 dimer inflexible thus incompatible to instigate triplex Tc-binding as observed in HCMV. While pp150 is essential in HCMV, our pp150-deletion mutant of MCMV remained viable though with attenuated infectivity and exhibiting defects in retaining viral genome. These results thus invalidate targeting pp150, but lend support to targeting capsid proteins, when using MCMV as a model for HCMV pathogenesis and therapeutic studies.

Murine Cytomegalovirus Protein pM91 Interacts with pM79 and Is Critical for Viral Late Gene Expression.

Viral gene expression is tightly regulated during cytomegalovirus (CMV) lytic replication, but the detailed mechanism of late gene transcription remains to be fully understood. Previous studies reported that six viral proteins (named viral transactivation factors [vTFs]) supporting late gene expression were conserved in beta- and gammaherpesviruses but not in alphaherpesviruses. Here, we performed coimmunoprecipitation experiments to elucidate the organization of these six proteins in murine CMV. Our results showed that these proteins formed a complex by both direct and indirect interactions. Specifically, pM91 strongly bound to pM79 even in the absence of other vTFs. Similar to pM79, pM91 exhibited early-late expression kinetics and localized within nuclear viral replication compartments during infection. Functional analysis was also performed using the pM91-deficient virus. Real-time PCR results revealed that abrogation of M91 expression markedly reduced viral late gene expression and progeny virus production without affecting viral DNA synthesis. Using mutagenesis, we found that residues E61, D62, D89, and D96 in pM91 were required for the pM91-pM79 interaction. Disruption of the interaction via E61A/D62A or D89A/D96A double mutation in the context of virus infection inhibited progeny virus production. Our data indicate that pM91 is a component of the viral late gene transcription factor complex and that the pM91-pM79 interaction is essential for viral late gene expression.IMPORTANCE Cytomegalovirus (CMV) infection is the leading cause of birth defects and causes morbidity and mortality in immunocompromised patients. The regulation of viral late gene transcription is not well elucidated, and understanding of this process benefits the development of novel therapeutics against CMV infection. This study (i) identified that six viral transactivation factors encoded by murine CMV form a complex, (ii) demonstrated that pM91 interacts with pM79 and that pM91 and pM79 colocalize in the nuclear viral replication compartments, (iii) confirmed that pM91 is critical for viral late gene expression but dispensable for viral DNA replication, and (iv) revealed that the pM91-pM79 interaction is required for progeny virus production. These findings give an explanation of how CMV regulates late gene expression and have important implications for the design of antiviral strategies.

Cytomegalovirus gp40/m152 Uses TMED10 as ER Anchor to Retain MHC Class I.

The murine cytomegalovirus immunoevasin m152/gp40 binds major histocompatibility complex (MHC) class I molecules and retains them in the early secretory pathway by a previously unknown mechanism, preventing antigen presentation to CD8+ T cells. We show that retention of class I and of gp40 itself depends on a lumenal linker sequence in gp40. With unbiased co-immunoprecipitation and mass spectrometry, we find that, through this linker, gp40 binds to TMED10/Tmp21/p24δ1, a member of the p24 family of endoplasmic reticulum (ER)/Golgi transmembrane proteins. We show that the C-terminal KKxxx Golgi-to-ER retrieval signal of TMED10 is required for gp40-mediated retention of class I. We thus identify a viral interaction partner of the p24 proteins and their exploitation for viral immune evasion.

Two glycosaminoglycan-binding domains of the mouse cytomegalovirus-encoded chemokine MCK-2 are critical for oligomerization of the full-length protein.

Chemokines are essential for antimicrobial host defenses and tissue repair. Herpesviruses and poxviruses also encode chemokines, copied from their hosts and repurposed for multiple functions, including immune evasion. The CC chemokine MCK-2 encoded by mouse CMV (MCMV) has an atypical structure consisting of a classic chemokine domain N-terminal to a second unique domain, resulting from the splicing of MCMV ORFs m131 and m129 MCK-2 is essential for full MCMV infectivity in macrophages and for persistent infection in the salivary gland. However, information about its mechanism of action and specific biochemical roles for the two domains has been lacking. Here, using genetic, chemical, and enzymatic analyses of multiple mouse cell lines as well as primary mouse fibroblasts from salivary gland and lung, we demonstrate that MCK-2 binds glycosaminoglycans (GAGs) with affinities in the following order: heparin > heparan sulfate > chondroitin sulfate = dermatan sulfate. Both MCK-2 domains bound these GAGs independently, and computational analysis together with site-directed mutagenesis identified five basic residues distributed across the N terminus and the 30s and 50s loops of the chemokine domain that are important GAG binding determinants. Both domains were required for GAG-dependent oligomerization of full-length MCK-2. Thus, MCK-2 is an atypical viral chemokine consisting of a CC chemokine domain and a unique non-chemokine domain, both of which bind GAGs and are critical for GAG-dependent oligomerization of the full-length protein.