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COVID-19 vaccines prevent severe forms of the disease, but do not warrant complete pro-tection against breakthrough infections. This could be due to suboptimal mucosal immu-nity at the site of virus entry, given that all currently approved vaccines are administeredvia the intramuscular route. In this study, we assessed humoral and cellular immuneresponses in BALB/c mice after intranasal and intramuscular immunization with adenovi-ral vector ChAdOx1-S expressing full- length Spike protein of SARS-CoV-2. We showed thatboth routes of vaccination induced a potent IgG antibody response, as well as robust neu- tralizing capacity, but intranasal vaccination elicited a superior IgA antibody titer in thesera and in the respiratory mucosa. Bronchoalveolar lavage from intranasally immunizedmice efficiently neutralized SARS-CoV-2, which has not been the case in intramuscularlyimmunized group. Moreover, substantially higher percentages of epitope- specific CD8 Tcells exhibiting a tissue resident phenotype were found in the lungs of intranasally immu-nized animals. Finally, both intranasal and intramuscular vaccination with ChAdOx1- Sefficiently protected the mice after the challenge with recombinant herpesvirus express-ing the Spike protein. Our results demonstrate that intranasal application of adenoviralvector ChAdOx1-S induces superior mucosal immunity and therefore could be a promis-ing strategy for putting the COVID-19 pandemic under control.

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 , 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 . 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. 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 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 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.

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.

Next Generation Sequencing allows for deep analysis of transcriptional activity in cells and tissues, however it is still a cost intensive method that demands well versed data handling. Reverse transcription quantitative PCR (RT-qPCR) is the most commonly used method to measure gene expression levels, however the information gathered is quite small in comparison to NGS. A newer method called nanoString allows for highly multiplexed gene expression analysis by detecting mRNAs without the use of enzymes for reverse transcription or amplification even for single cells or low input material. The method can be done in 1.5 days and data are quickly analyzed by the accompanied user friendly software. Our aim was to investigate this new method and compare it to the existing alternatives, while investigating murine Cytomegalovirus (mCMV) infection and latency.mCMV infected murine embryonic fibroblasts (MEF), lung and salivary glands from BALB/c mice were evaluated at different stages of infection. A set of 30 custom designed nanoString probes were tested, 20 probes specific for mCMV genes, 6 probes for host genes known to be influenced by viral infection and 4 reference gene specific probes. nanoString counts were compared to published RNA-Seq RPKM.We found that nanoString can be used for analysis of cytomegalovirus gene expression during acute infection in vitro and in vivo, both for virus specific and host genes. Although some transcripts show different expression rates in comparison to NGS data, the most abundant transcripts are comparable. When tissues are infected, there are significantly fewer transcripts than in MEFs, and consistent with previous work there are significant differences in relevant abundance between MEF and tissues. We were unable to detect our viral transcripts of interest in latently infected tissue.For viruses with annotated transcriptomes, nanoString allows simultaneous quantitation of multiple virus and host genes. One huge advantage of the platform is rapid turnaround and simplicity of analysis. It should prove to be very useful to explore host virus interactions during acute infection, but it is unclear if it has adequate sensitivity for analysis during latency in immunocompetent mice.

Using a mouse model of congenital cytomegalovirus infection, evidence is provided for a pathogenic role of NK and ILC1 cells in the brain. Although brain-infiltrating innate immune cells fail to control infection, they orchestrate pathological inflammatory responses that lead to altered cerebellar development., Congenital human cytomegalovirus (cHCMV) infection of the brain is associated with a wide range of neurocognitive sequelae. Using infection of newborn mice with mouse cytomegalovirus (MCMV) as a reliable model that recapitulates many aspects of cHCMV infection, including disseminated infection, CNS infection, altered neurodevelopment, and sensorineural hearing loss, we have previously shown that mitigation of inflammation prevented alterations in cerebellar development, suggesting that host inflammatory factors are key drivers of neurodevelopmental defects. Here, we show that MCMV infection causes a dramatic increase in the expression of the microglia-derived chemokines CXCL9/CXCL10, which recruit NK and ILC1 cells into the brain in a CXCR3-dependent manner. Surprisingly, brain-infiltrating innate immune cells not only were unable to control virus infection in the brain but also orchestrated pathological inflammatory responses, which lead to delays in cerebellar morphogenesis. Our results identify NK and ILC1 cells as the major mediators of immunopathology in response to virus infection in the developing CNS, which can be prevented by anti–IFN-γ antibodies., Graphical Abstract

Human cytomegalovirus (HCMV) is a leading viral cause of congenital infections in the central nervous system (CNS) and may result in severe long-term sequelae. High rates of sequelae following congenital HCMV infection and insufficient antiviral therapy in the perinatal period makes the development of an HCMV-specific vaccine a high priority of modern medicine. Due to the species specificity of HCMV, animal models are frequently used to study CMV pathogenesis. Studies of murine cytomegalovirus (MCMV) infections of adult mice have played a significant role as a model of CMV biology and pathogenesis, while MCMV infection of newborn mice has been successfully used as a model of perinatal CMV infection. Newborn mice infected with MCMV have high levels of viremia during which the virus establishes a productive infection in most organs, coupled with a robust inflammatory response. Productive infection in the brain parenchyma during early postnatal period leads to an extensive nonnecrotizing multifocal widespread encephalitis characterized by infiltration of components of both innate and adaptive immunity. As a result, impairment in postnatal development of mouse cerebellum leads to long-term motor and sensor disabilities. This chapter summarizes current findings of rodent models of perinatal CMV infection and describes methods for analysis of perinatal MCMV infection in newborn mice.

Viral infections during pregnancy are a considerable cause of adverse outcomes and birth defects, and the underlying mechanisms are poorly understood. Among those, cytomegalovirus (CMV) infection stands out as the most common intrauterine infection in humans, putatively causing early pregnancy loss. We employed murine CMV as a model to study the consequences of viral infection on pregnancy outcome and fertility maintenance. Even though pregnant mice successfully controlled CMV infection, we observed highly selective, strong infection of corpus luteum (CL) cells in their ovaries. High infection densities indicated complete failure of immune control in CL cells, resulting in progesterone insufficiency and pregnancy loss. An abundance of gap junctions, absence of vasculature, strong type I interferon (IFN) responses, and interaction of innate immune cells fully protected the ovarian follicles from viral infection. Our work provides fundamental insights into the effect of CMV infection on pregnancy loss and mechanisms protecting fertility.

The cloning of herpesviruses as bacterial artificial chromosomes (BACs) has revolutionized the study of herpesvirus biology, allowing rapid and precise manipulation of viral genomes. Several clinical strains of human cytomegalovirus (HCMV) have been cloned as BACs; however, no low-passage strains of murine CMV (MCMV), which provide a model mimicking these isolates, have been cloned. Here, the low-passage G4 strain of was BAC cloned. G4 carries an m157 gene that does not ligate the natural killer (NK) cell-activating receptor, Ly49H, meaning that unlike laboratory strains of MCMV, this virus replicates well in C57BL/6 mice. This BAC clone exhibited normal replication during acute infection in the spleen and liver but was attenuated for salivary gland tropism. Next-generation sequencing revealed a C-to-A mutation at nucleotide position 188422, located in the 3′ untranslated region of sgg1, a spliced gene critical for salivary gland tropism. Repair of this mutation restored tropism for the salivary glands. Transcriptional analysis revealed a novel spliced gene within the sgg1 locus. This small open reading frame (ORF), sgg1.1, starts at the 3′ end of the first exon of sgg1 and extends exon 2 of sgg1. This shorter spliced gene is prematurely terminated by the nonsense mutation at nt 188422. Sequence analysis of tissue culture-passaged virus demonstrated that sgg1.1 was stable, although other mutational hot spots were identified. The G4 BAC will allow studies in a broader range of mice, avoiding the strong NK cell responses seen in B6 mice with other MCMV BAC-derived MCMVs. Murine cytomegalovirus (MCMV) is widely used as a model of human CMV (HCMV) infection. However, this model relies on strains of MCMV that have been serially passaged in the laboratory for over four decades. These laboratory strains have been cloned as bacterial artificial chromosomes (BACs), which permits rapid and precise manipulation. Low-passage strains of MCMV add to the utility of the mouse model of HCMV infection but do not exist as cloned BACs. This study describes the first such low-passage MCMV BAC. This BAC-derived G4 was initially attenuated , with subsequent full genomic sequencing revealing a novel spliced transcript required for salivary gland tropism. These data suggest that MCMV, like HCMV, undergoes tissue culture adaptation that can limit growth and supports the use of BAC clones as a way of standardizing viral strains and minimizing interlaboratory strain variation.

The rapid activation of pattern recognition receptor (PRR)-mediated type I interferon (IFN) signaling is crucial for the host response to infection. In turn, human cytomegalovirus (HCMV) must evade this potent response to establish life-long infection. Here, we reveal that the HCMV tegument protein UL35 antagonizes the activation of type I IFN transcription downstream of the DNA and RNA sensors cGAS and RIG-I, respectively. We show that ectopic expression of UL35 diminishes the type I IFN response, while infection with a recombinant HCMV lacking UL35 induces an elevated type I IFN response compared to wildtype HCMV. With a series of luciferase reporter assays and the analysis of signaling kinetics upon HCMV infection, we observed that UL35 downmodulates PRR signaling at the level of the key signaling factor TANK-binding kinase 1 (TBK1). Finally, we demonstrate that UL35 and TBK1 co-immunoprecipitate when co-expressed in HEK293T cells. In addition, we show that a previously reported cellular binding partner of UL35, O-GlcNAc transferase (OGT), post-translationally GlcNAcylates UL35, but that this modification is not required for the antagonizing effect of UL35 on PRR signaling. In summary, we have identified UL35 as the first HCMV protein to antagonize the type I IFN response at the level of TBK1, thereby enriching our understanding of how this important herpesvirus escapes host immune responses.

Cytomegaloviruses (CMVs) are ubiquitous pathogens known to employ numerous immunoevasive strategies that significantly impair the ability of the immune system to eliminate the infected cells. Here, we report that the single mouse CMV (MCMV) protein, m154, downregulates multiple surface molecules involved in the activation and costimulation of the immune cells. We demonstrate that m154 uses its cytoplasmic tail motif, DD, to interfere with the adaptor protein-1 (AP-1) complex, implicated in intracellular protein sorting and packaging. As a consequence of the perturbed AP-1 sorting, m154 promotes lysosomal degradation of several proteins involved in T cell costimulation, thus impairing virus-specific CD8+ T cell response and virus control in vivo. Additionally, we show that HCMV infection similarly interferes with the AP-1 complex. Altogether, we identify the robust mechanism employed by single viral immunomodulatory protein targeting a broad spectrum of cell surface molecules involved in the antiviral immune response.

Murine cytomegalovirus (MCMV) initiates the stepwise establishment of the pre-assembly compartment (pre-AC) in the early phase of infection by the expansion of the early endosome (EE)/endosomal recycling compartment (ERC) interface and relocation of the Golgi complex. We depleted Vps34-derived phosphatidylinositol-3-phosphate (PI(3)P) at EEs by VPS34-IN1 and inhibited PI(3)P-associated functions by overexpression of 2xFYVE- and p40PX PI(3)P-binding modules to assess the role of PI(3)P-dependent EE domains in the pre-AC biogenesis. We monitored the accumulation of Rab10 and Evectin-2 in the inner pre-AC and the relocation of GM130-positive cis-Golgi organelles to the outer pre-AC by confocal microscopy. Although PI(3)P- and Vps34-positive endosomes build a substantial part of pre-AC, the PI(3)P depletion and the inhibition of PI(3)P-associated functions did not prevent the establishment of infection and progression through the early phase. The PI(3)P depletion in uninfected and MCMV-infected cells rapidly dispersed PI(3)P-bond proteins and reorganized EEs, including ablation of EE-to-ERC transport and relocation of Rab11 endosomes. The PI(3)P depletion one hour before pre-AC initiation and overexpression of 2xFYVE and p40PX domains neither prevented Rab10- and Evectin-2 accumulation, nor Golgi unlinking and relocation. These data demonstrate that PI(3)P-dependent functions, including the Rab11-dependent EE-to-ERC route, are dispensable for pre-AC initiation. Nevertheless, the virus growth was drastically reduced in PI(3)P-depleted cells, indicating that PI(3)P-associated functions are essential for the late phase of infection.

Železnjak et al. demonstrate that two MCMV-encoded proteins interact with MHC I molecules, forming an altered-self complex that prevents missing self recognition by increasing specificity for inhibitory Ly49 receptors. This led to the evolution of CMV-specific activating Ly49s., 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., Graphical Abstract

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

NK cells play an important role in the control of viral infections. Cytomegaloviruses have played a big part in the accumulation of current knowledge describing how NK cells recognize and eliminate infected cells. The interference with these functions at multiple levels may not only play a role in the control of primary infections or reactivations but can also impact other arms of the immune system and leave a long-lasting stable imprint on the NK cell population. These imprints may, in turn, modify how we respond to other infections. Understanding these processes will allow us to design better diagnostic approaches and new treatment options through manipulation of our immune responses and the viruses themselves.

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

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

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

Contains fulltext : 171662.pdf (Publisher’s version ) (Open Access) Antibodies are widely exploited as research/diagnostic tools and therapeutics. Despite providing exciting research opportunities, the multitude of available antibodies also offers a bewildering array of choice. Importantly, not all companies comply with the highest standards, and thus many reagents fail basic validation tests. The responsibility for antibodies being fit for purpose rests, surprisingly, with their user. This paper condenses the extensive experience of the European Monoclonal Antibody Network to help researchers identify antibodies specific for their target antigen. A stepwise strategy is provided for prioritising antibodies and making informed decisions regarding further essential validation requirements. Web-based antibody validation guides provide practical approaches for testing antibody activity and specificity. We aim to enable researchers with little or no prior experience of antibody characterization to understand how to determine the suitability of their antibody for its intended purpose, enabling both time and cost effective generation of high quality antibody-based data fit for publication.

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

Cytomegaloviruses (CMVs) are highly prevalent, persistent human pathogens that not only evade but also shape our immune responses. Natural killer (NK) cells play an important role in the control of CMV and CMVs have in turn developed a plethora of immunoevasion mechanisms targeting NK cells. This complex interplay can leave a long-lasting imprint on the immune system in general and affect responses toward other pathogens and vaccines. This review aims to provide an overview of NK cell biology and development, the manipulation of NK cells by CMVs and the potential impact of these evasion strategies on responses to vaccination.

Cytomegaloviruses (CMVs) have dedicated a large portion of their genome towards immune evasion targeting many aspects of the host immune system, particularly NK cells. However, the host managed to cope with the infection by developing multiple mechanisms to recognize viral threat and counterattack it, thus illustrating never-ending evolutionary interplay between CMV and its host. In this review, we will focus on several mechanisms of NK cell evasion by mouse CMV (MCMV), the role of host inhibitory and activating Ly49 receptors involved in the virus control and acquisition of adaptive features by NK cells as a consequence of MCMV infection.

Jonjic et al. show that inflammatory macrophages play an essential role in the control of murine CMV (MCMV) infection through a DNAM-1–PVR pathway., The poliovirus receptor (PVR) is a ubiquitously expressed glycoprotein involved in cellular adhesion and immune response. It engages the activating receptor DNAX accessory molecule (DNAM)-1, the inhibitory receptor TIGIT, and the CD96 receptor with both activating and inhibitory functions. Human cytomegalovirus (HCMV) down-regulates PVR expression, but the significance of this viral function in vivo remains unknown. Here, we demonstrate that mouse CMV (MCMV) also down-regulates the surface PVR. The m20.1 protein of MCMV retains PVR in the endoplasmic reticulum and promotes its degradation. A MCMV mutant lacking the PVR inhibitor was attenuated in normal mice but not in mice lacking DNAM-1. This attenuation was partially reversed by NK cell depletion, whereas the simultaneous depletion of mononuclear phagocytes abolished the virus control. This effect was associated with the increased expression of DNAM-1, whereas TIGIT and CD96 were absent on these cells. An increased level of proinflammatory cytokines in sera of mice infected with the virus lacking the m20.1 and an increased production of iNOS by inflammatory monocytes was observed. Blocking of CCL2 or the inhibition of iNOS significantly increased titer of the virus lacking m20.1. In this study, we have demonstrated that inflammatory monocytes, together with NK cells, are essential in the early control of CMV through the DNAM-1–PVR pathway.

The global transcriptional program of murine cytomegalovirus (MCMV), involving coding, noncoding, and antisense transcription, remains unknown. Here we report an oligonucleotide custom microarray platform capable of measuring both coding and noncoding transcription on a genome-wide scale. By profiling MCMV wild-type and immediate-early mutant strains in fibroblasts, we found rapid activation of the transcriptome by 6.5 h postinfection, with absolute dependency on ie3 , but not ie1 or ie2 , for genomic programming of viral gene expression. Evidence is also presented to show, for the first time, genome-wide noncoding and bidirectional transcription at late stages of MCMV infection.

Galectin-3, a β galactoside–binding lectin, plays an important role in the processes relevant to tumorigenesis such as malignant cell transformation, invasion and metastasis. We have investigated whether deletion of Galectin-3 in the host affects the metastasis of B16F1 malignant melanoma. Galectin-3-deficient (Gal-3−/−) mice are more resistant to metastatic malignant melanoma as evaluated by number and size of metastatic colonies in the lung. In vitro assays showed lower number of attached malignant cells in the tissue section derived from Gal-3−/− mice. Furthermore, lack of Galectin-3 correlates with higher serum levels of IFN-γ and IL-17 in tumor bearing hosts. Interestingly, spleens of Gal-3−/− mice have lower number of Foxp3+ T cells after injection of B16F1 melanoma cells. Finally, we found that while CD8+ T cell and adherent cell cytotoxicity were similar, there was greater cytotoxic activity of splenic NK cells of Gal-3−/− mice compared with “wild-type” (Gal-3 +/+ ) mice. Despite the reduction in total number of CD3e−NK1.1+, Gal-3−/− mice constitutively have a significantly higher percentage of effective cytotoxic CD27highCD11bhigh NK cells as well as the percentage of immature CD27highCD11blow NK cells. In contrast, CD27lowCD11bhigh less functionally exhausted NK cells and NK cells bearing inhibitory KLRG1 receptor were more numerous in Gal-3 +/+ mice. It appears that lack of Galectin-3 affects tumor metastasis by at least two independent mechanisms: by a decrease in binding of melanoma cells onto target tissue and by enhanced NK-mediated anti-tumor response suggesting that Galectin-3 may be considered as therapeutic target.

Since their discovery, our understanding of NK cells has evolved from branding them marginal innate immunity cells to key players in anti-viral and anti-tumor immunity. Importance of NK cells in control of various viral infections is perhaps best illustrated by the existence of plethora of viral mechanisms aimed to modulate their function. These mechanisms include not only virally encoded immunoevasion proteins but also viral miRNA. Moreover, the evidence has been accumulated supporting the role of viral immunoevasion of NK cells in viral pathogenesis in vivo.

Recent advances in sequencing technology enabled us to perform comprehensive transcriptomic profiling of MCMV infection of MEFs and demonstrate that transcriptional profile and coding potential of herpesviruses have a much greater complexity than previously anticipated. Our analysis identified numerous novel spliced and unspliced MCMV transcripts, among which the most abundant viral transcripts are novel transcripts of unknown function. Among these, MAT (Most Abundant Transcript) is the most highly expressed throughout the infection. It has been shown earlier that MAT encodes a protein (Juranic Lisnic et al (2013) PLOS Pathogens) and contains a binding site for cellular microRNA miR-27 (Marcinowski et al (2012) PLOS Pathogens), making it a first reported viral transcript with coding and non-coding functions. We have recently detected an additional protein encoded by this enigmatic transcript, and here we report that deletion of the MCMV genomic region encoding MAT results in attenuation of the virus in vivo. However, the underlying mechanisms are not completely elucidated due to multiple functions MAT performs in the infected cells. On one hand, MAT deficiency results in failure to deplete cellular levels of miR-27, an important regulator of cell-cycle, while on the other MAT deletion mutants evade the detection by NK cells through activating Ly49 receptors P, L and D2. We also show that absence of MAT results in dramatic changes of host and viral peptides presented in the context of MHC I. In conclusion, not only does MAT provide an excellent example of the complex regulatory potential of herpesviral genes, where single transcripts exerts numerous and diverse functions, it also raises the question of how many new herpesviral genes, especially immune evasion genes, remain to be discovered.

Human cytomegalovirus (HCMV) is a leading viral cause of congenital infections in the central nervous system (CNS) and may result in severe long-term sequelae. High rates of sequelae following congenital HCMV infection and insufficient antiviral therapy in the perinatal period makes the development of an HCMV-specific vaccine a high priority of modern medicine. Due to the species specificity of HCMV, animal models are frequently used to study CMV pathogenesis. Studies of murine cytomegalovirus (MCMV) infections of adult mice have played a significant role as a model of CMV biology and pathogenesis, while MCMV infection of newborn mice has been successfully used as a model of perinatal CMV infection. Newborn mice infected with MCMV have high levels of viremia during which the virus establishes a productive infection in most organs, coupled with a robust inflammatory response. Productive infection in the brain parenchyma during early postnatal period leads to an extensive nonnecrotizing multifocal widespread encephalitis characterized by infiltration of components of both innate and adaptive immunity. As a result, impairment in postnatal development of mouse cerebellum leads to long-term motor and sensor disabilities. This chapter summarizes current findings of rodent models of perinatal CMV infection and describes methods for analysis of perinatal MCMV infection in newborn mice.

Major gaps in our knowledge of pathogen genes and how these gene products interact with host gene products to cause disease represent a major obstacle to progress in vaccine and antiviral drug development for the herpesviruses. To begin to bridge these gaps, we conducted a dual analysis of Murine Cytomegalovirus (MCMV) and host cell transcriptomes during lytic infection. We analyzed the MCMV transcriptome during lytic infection using both classical cDNA cloning and sequencing of viral transcripts and next generation sequencing of transcripts (RNA-Seq). We also investigated the host transcriptome using RNA-Seq combined with differential gene expression analysis, biological pathway analysis, and gene ontology analysis. We identify numerous novel spliced and unspliced transcripts of MCMV. Unexpectedly, the most abundantly transcribed viral genes are of unknown function. We found that the most abundant viral transcript, recently identified as a noncoding RNA regulating cellular microRNAs, also codes for a novel protein. To our knowledge, this is the first viral transcript that functions both as a noncoding RNA and an mRNA. We also report that lytic infection elicits a profound cellular response in fibroblasts. Highly upregulated and induced host genes included those involved in inflammation and immunity, but also many unexpected transcription factors and host genes related to development and differentiation. Many top downregulated and repressed genes are associated with functions whose roles in infection are obscure, including host long intergenic noncoding RNAs, antisense RNAs or small nucleolar RNAs. Correspondingly, many differentially expressed genes cluster in biological pathways that may shed new light on cytomegalovirus pathogenesis. Together, these findings provide new insights into the molecular warfare at the virus-host interface and suggest new areas of research to advance the understanding and treatment of cytomegalovirus-associated diseases., Author Summary We have conducted a comprehensive analysis of the murine cytomegalovirus and host cell transcriptomes during lytic infection. We identify numerous novel spliced and unspliced transcripts of MCMV. Unexpectedly, the most abundantly transcribed viral genes are of unknown function. We found that the most abundant viral transcript, recently identified as a noncoding RNA regulating cellular microRNAs, also codes for a novel protein. To our knowledge, this is the first viral transcript that functions both as a noncoding RNA and an mRNA. Infection alters expression of many unexpected host genes, including many noncoding RNA genes. Correspondingly, many cluster in unexpected biological pathways that may shed new light on cytomegalovirus pathogenesis. Together, these findings provide new insights into the molecular warfare at the virus-host interface and suggest new areas of research to advance the understanding and treatment of cytomegalovirus-associated diseases.

Human cytomegalovirus (HCMV) is a ubiquitous human pathogen responsible for devastating congenital disease and life-threatening complications in immune-suppressed patients. Available treatments have many shortcomings and effective vaccine is still lacking. Major obstacles to progress in vaccine and antiviral drug development are (1) species specificity of HCMV, and (2) gaps in our understanding of viral genes and their interaction with host genes. First limitation is circumvented by the use of model animal viruses, especially murine cytomegalovirus (MCMV). We sought to alleviate the second problem by studying MCMV transcriptome using two approaches: classical cDNA library analysis and next generation sequencing (RNASeq). This dual analysis revealed incredible complexity of MCMV transcriptome, detected numerous novel viral spliced and unspliced transcripts as well as transcription from intergenic regions, and showed that expression levels of viral transcripts vary by several orders of magnitude. Unexpectedly, most top expressed genes were of unknown functions and were improperly annotated. Therefore, this analysis provides the first comprehensive overview of MCMV transcriptome, underscores the necessity of transcriptomic analyses in providing evidence-based genome annotation and could serve as the first step towards re-annotation of MCMV genome. The most abundant viral transcript, recently identified as a noncoding RNA regulating cellular microRNAs [18, 84], was shown to also code for a novel protein(s). This is the first viral transcript that functions both as a noncoding RNA and an mRNA. In this work it is also shown that this transcript’s 5’ UTR plays a role in NK cell recognition of infected cells via activating Ly49 receptors. Analysis of host transcriptome showed that lytic infection revealed that many unexpected gene groups are disregulated in response to the infection. Such systematic analysis may shed new light on cytomegalovirus pathogenesis and suggests new areas of research.

Cytomegaloviruses express large amounts of viral miRNAs during lytic infection, yet, they only modestly alter the cellular miRNA profile. The most prominent alteration upon lytic murine cytomegalovirus (MCMV) infection is the rapid degradation of the cellular miR-27a and miR-27b. Here, we report that this regulation is mediated by the ∼1.7 kb spliced and highly abundant MCMV m169 transcript. Specificity to miR-27a/b is mediated by a single, apparently optimized, miRNA binding site located in its 3′-UTR. This site is easily and efficiently retargeted to other cellular and viral miRNAs by target site replacement. Expression of the 3′-UTR of m169 by an adenoviral vector was sufficient to mediate its function, indicating that no other viral factors are essential in this process. Degradation of miR-27a/b was found to be accompanied by 3′-tailing and -trimming. Despite its dramatic effect on miRNA stability, we found this interaction to be mutual, indicating potential regulation of m169 by miR-27a/b. Most interestingly, three mutant viruses no longer able to target miR-27a/b, either due to miRNA target site disruption or target site replacement, showed significant attenuation in multiple organs as early as 4 days post infection, indicating that degradation of miR-27a/b is important for efficient MCMV replication in vivo., Author Summary MicroRNAs are small, non-coding RNAs which shape and fine-tune gene expression of at least a third of our genes. During millions of years of coevolution with their hosts, herpesviruses have both usurped the host cell miRNA machinery by expressing their own sets of miRNAs, and learned to modify host miRNA expression for their own needs. Recently, we reported on the rapid degradation of two cellular miRNAs upon lytic murine cytomegalovirus (MCMV) infection, namely miR-27a and miR-27b. In this paper, we show that their regulation is mediated by the highly abundant viral transcript m169. It targets miR-27a/b via a single binding site in its 3′-UTR, which can be efficiently retargeted to other cellular and viral miRNAs, enabling the efficient knock-down of individual miRNAs of interest. Degradation of miR-27a/b is preceded by its 3′-tailing and -trimming. Most interestingly, three mutant viruses unable to target miR-27a/b showed significantly lower virus titers in various organs during acute MCMV infection, indicating that degradation of miR-27a/b is important for efficient virus replication in vivo.

Human cytomegalovirus (HCMV) is an important human pathogen, infecting over 70% of human population and responsible for devastating congenital disease and organ transplant failure in immunologically immature or suppressed patients. Unfortunately, it infects only humans and is therefore hard to study. Murine cytomegalovirus (MCMV) is the most widely used model to study HCMV) pathogenesis in vivo since it shares many biological and pathological features and is genetically related to HCMV. Functions of many of its genes, especially immunoevasins, have been characterized thanks to plethora of deletion mutants. Although generation of deletion mutants relies on genomic maps, our currently used map of MCMV genome has many drawbacks. Current annotations were constructed using bioinformatics tools to predict ORFs with verification by genomic tilling arrays. Such analyses are poorly adapted to condensed viral genomes that contain many overlapping genes and can therefore miss ORFs or rare alternative splicing sites. With this in mind we have set to analyze the transcriptome of MCMV using two different approaches: cDNA library and deep sequencing. Based on 448 cDNA clones and deep sequencing data we have constructed a map of MCMV transcriptome which differs significantly with currently used annotations. We have detected multiple instances where transcriptional products differed from predicted ORFs (longer or shorter, transcripts overlapping several predicted ORFs), new splice variants, transcripts in regions annotated as non-coding and coding regions with very low transcription. While we have detected several antisense transcripts, the level of antisense transcription was significantly lower than was observed for HCMV. Surprisingly, one transcript dominated MCMV transcriptome comprising over 1/3 of all transcripts. Despite its high abundance, its function was unknown. 1.7 kb in length, most abundant transcript (MAT) overlaps predicted m168 in antisense and m169 in sense orientation. We have previously observed that this region of MCMV genome is essential for activating Ly49 (P, L and D2) recognition of m04/MHC I complexes. We have shown that MAT transcript encodes poorly translated 17 kDa protein of unknown function and contains binding site for cellular microRNA miR-27 in its 3’-UTR leading to its degradation. Due to MATs abundance, it effectively depletes cellular levels of miR-27 upon infection. To our knowledge MAT is the first transcript described that contains both coding and non-coding functions. Interestingly neither function of miR sponge nor the produced protein are responsible for recognition by activating Ly49 receptors but 5’-UTR. Mechanisms by which 5’UTR mediates activating Ly49 recognition is currently being investigated.

The early detection of herpesvirus infection by pattern recognition receptors (PRRs) triggers the production of type I interferons (IFN), which exert potent antiviral effects. Whilst MCMV is recognized to induce strong innate immune responses, UV inactivation of MCMV results in an enhanced type I IFN response, indicating that the virus actively downmodulates innate immune signaling. Using an unbiased luciferase-based assay to screen an MCMV cDNA library, we have identified the M35 protein as a negative regulator of PRR-dependent type I IFN signaling. Infection of primary bone marrow derived macrophages with an MCMV mutant lacking M35 (M35stop) results in increased secretion of type I IFN as compared to wild-type (WT) MCMV. To confirm the physiological role of M35, we infected BALB/c mice with MCMV M35stop to assess the subsequent type I IFN response. MCMV M35stop induces significantly higher type I IFN serum levels than WT MCMV 8 h post infection, which correlates well with expression of tegument M35 protein. MCMV M35stop replicates to significantly reduced titres compared to WT MCMV in the spleen at day 3 post infection. MCMV is then disseminated via secondary viremia to the salivary glands, a site of persistence and dissemination for MCMV. MCMV M35stop is undetectable from the salivary glands from as early as 7 days post infection. This exclusion of M35stop from the salivary glands indicates that M35 is a critical determinant of viral replication in vivo. Collectively, our data demonstrate the first characterisation of M35 as a novel antagonist of type I IFN signaling downstream of PRRs.

Despite its ubiquity the pathogenesis of HCMV infection is still poorly understood and currently no effective vaccine is available. Murine cytomegalovirus (MCMV) is biologically similar and genetically related to HCMV and is therefore considered to be an excellent model for in vivo studies. To date, many new immunoevasins have been characterized thanks to genetic approaches using gene deletion mutant strains. However, the current genomic map of MCMV relies heavily on open reading frames (ORFs) predicted using computational methods and, more recently, using genomic tilling arrays. Such analyses can miss certain ORFs, spliced transcripts and do not give information about the exact 5’ and 3’ ends of the transcripts. Therefore, the goal of this project was to characterize all of the viral transcription products that accumulate in infected cells, including both polyadenylated and non-polyadenylated transcripts. Similar transcriptomic analysis of polyadenylated transcripts in HCMV revealed many novel genes and gene products, and our analysis of MCMV’s transcriptome, reveals unexpected complexity of the MCMV genome which could not have been envisioned with ORF prediction software alone. We have detected a number of new transcripts and novel splice-variants, along with several S-AS pairs. Currently we’re employing deep sequencing to complement our cDNA library analysis and to extend our analysis to viral and mouse transcription profiles in vivo using WT virus and our published recombinant virus harboring the murine Rae1-gamma gene, which codes for a ligand of the activating natural killer cell receptor, NKG2D. In collaboration with IT researchers all of this data is integrated with bioinformatic tools in a publicly accessible database of the CMV transcriptome.

Human CMV (HCMV) is a major cause of morbidity and mortality in both congenitally infected and immunocompromised individuals. Development of an effective HCMV vaccine would help protect these vulnerable groups. NK group 2, member D (NKG2D) is a potent activating receptor expressed by cells of the innate and adaptive immune systems. Its importance in HCMV immune surveillance is indicated by the elaborative evasion mechanisms evolved by the virus to avoid NKG2D. In order to study this signaling pathway, we engineered a recombinant mouse CMV expressing the high-affinity NKG2D ligand RAE-1γ (RAE-1γMCMV). Expression of RAE-1γ by MCMV resulted in profound virus attenuation in vivo and lower latent viral DNA loads. RAE-1γMCMV infection was efficiently controlled by immunodeficient hosts, including mice lacking type I interferon receptors or immunosuppressed by sublethal γ-irradiation. Features of MCMV infection in neonates were also diminished. Despite tight innate immune control, RAE-1γMCMV infection elicited strong and long-lasting protective immunity. Maternal RAE-1γMCMV immunization protected neonatal mice from MCMV disease via placental transfer of antiviral Abs. Despite strong selective pressure, the RAE-1γ transgene did not exhibit sequence variation following infection. Together, our results indicate that use of a recombinant virus encoding the ligand for an activating NK cell receptor could be a powerful approach to developing a safe and immunogenic HCMV vaccine.