CMV and natural killer cells: shaping the response to vaccination.

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

Murine CMV Expressing the High Affinity NKG2D Ligand MULT-1: A Model for the Development of Cytomegalovirus-Based Vaccines

The development of a vaccine against human cytomegalovirus (CMV) has been a subject of long-term medical interest. The research during recent years identified CMV as an attractive vaccine vector against infectious diseases and tumors. The immune response to CMV persists over a lifetime and its unique feature is the inflationary T cell response to certain viral epitopes. CMV encodes numerous genes involved in immunoevasion, which are non-essential for virus growth in vitro. The deletion of those genes results in virus attenuation in vivo, which enables us to dramatically manipulate its virulence and the immune response. We have previously shown that the murine CMV (MCMV) expressing RAE-1γ, one of the cellular ligands for the NKG2D receptor, is highly attenuated in vivo but retains the ability to induce a strong CD8+ T cell response. Here, we demonstrate that recombinant MCMV expressing high affinity NKG2D ligand murine UL16 binding protein-like transcript (MULT-1) (MULT-1MCMV) inserted in the place of its viral inhibitor is dramatically attenuated in vivo in a NK cell-dependent manner, both in immunocompetent adult mice and in immunologically immature newborns. MULT-1MCMV was more attenuated than the recombinant virus expressing RAE-1γ. Despite the drastic sensitivity to innate immune control, MULT-1MCMV induced an efficient CD8+ T cell response to viral and vectored antigens. By using in vitro assay, we showed that similar to RAE-1γMCMV, MULT-1 expressing virus provided strong priming of CD8+ T cells. Moreover, MULT-1MCMV was able to induce anti-viral antibodies, which after passing the transplacental barrier protect offspring of immunized mothers from challenge infection. Altogether, this study further supports the concept that CMV expressing NKG2D ligand possesses excellent characteristics to serve as a vaccine or vaccine vector

The Cytomegalovirus Tegument Protein UL35 Antagonizes Pattern Recognition Receptor-Mediated Type I IFN Transcription

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

The herpesviral antagonist m152 reveals differential activation of STING‐dependent IRF and NF‐κB signaling and STING's dual role during MCMV infection

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

The murine cytomegalovirus M35 protein antagonizes type I IFN induction downstream of pattern recognition receptors by targeting NF-κB mediated transcription.

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

Inflammatory monocytes and NK cells play a crucial role in DNAM-1-dependent control of cytomegalovirus infection

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 TIG IT, 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 TIG IT 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 pathwa

Modulation of natural killer cell activity by viruses

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

Analiza transkriptoma mišijeg citomegalovirusa

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.Svrha istraživanja Humani citomegalovirus široko je rasprostranjen patogen, a posebno je opasan za trudnice, novorođenčad i imunosuprimirane pacijente. Nažalost, efikasnog cjepivo nema, a postoji potreba i za efikasnijim i manje toksičnim antiviralnim lijekovima. Glavne prepreke razvoju novih antiviralnih ljekova i cjepiva jesu: (1) specifičnost za vrstu i (2) praznine u našem znanju i razumjevanju virusnih gena, interakcijama virusnih gena i domaćina te kako te interakcije izazivaju bolest. Prva prepreka uspješno se nadvladava korištenjem animalnih virusa, posebice mišjeg citomegalovirusa (MCMV). S ciljem nadvladavanja druge prepreke u ovom je radu provedena detaljna analiza transkriptoma mišjeg citomegalovirusa (MCMV) te analiza transkriptoma stanica domaćina tijekom litičke infekcije citomegalovirusom. Materijali i metode Transkriptom MCMV analiziran je na dva načina: klasičnom analizom cDNK knjižnice i sekvencioniranjem dobivenih klonova te analizom transkriptoma uz pomoć sekvencioniranja nove generacije (odnosno RNK-sekvencioniranjem, eng. RNASeq) koja omogućava paralelno praćenje i transkriptoma domaćina uz transkriptom virusa. Analiza transkriptoma domaćina rezultira vrlo dugačkim listama diferencijalno reguliranih gena iz kojih je teško izvući neko biološko značenje. Stoga su liste diferencijalno reguliranih gena domaćina podvrgnute analizi termina genske ontologije (eng. gene ontology analysis odnosno GO analiza) i analizom dereguliranih bioloških puteva. Transkripcijski kompleksne regije genoma MCMV dodatno su analizirane Northern hibridizacijom i metodom RT-PCR dok je korelacija između količine transkripata i proteina odabranih gena analizirana metodom Western blot. Na kraju, funkcija novog, prekrojenog transkripta MAT (most abundant transcript; najzastupljeniji transkript) analizirana je uz pomoć reporterskih stanica koje nose aktivacijske Ly49 receptore. Rezultati Ovaj rad predstavlja prvu detaljnu analizu transkriptoma MCMV-a korištenjem komplementarnih metoda analize cDNK knjižnice i RNASeq analizom, a rezultirala je identifikacijom brojnih novih transkripata MCMV, uključujući i nove prekrojene transkripte kao i transkripte koji se prepisuju sa intergenskih regija. Ustanovljeno je da najjače izraženi virusni transkripti imaju nepoznatu funkciju i često su pogrešno anotirani. Najjače izražen transkript (tzv. MAT transkript) prvi je virusni transkript koji ima i kodirajuću i nekodirajuću funkciju. Naime, nedavno je pokazano da se na njegovom 3' netranslatiranom kraju (3' UTR, od eng. 3' untranslated region) nalazi vezno mjesto za staničnu mikro-RNK [18, 84], dok je u ovom radu pokazano da on također kodira i barem još dva proteina. Uz ove dvije navedene funkcije, u ovom je radu otkriveno da je 5' netranslatirani kraj (5'UTR) MAT transkripta bitan virusni faktor kojeg na inificranim stanicama prepoznaju stanice prirodne ubojice pomoću aktivacijskih receptora Ly49. Analiza transkriptoma stanica domaćina pokazala je da litička infekcija virusom MCMV izaziva izrazite promjene u ekspresijskom profilu gena domaćina: ekspresija gotovo trećine gena miša promijenila se uslijed infekcije virusom MCMV. Geni čija se ekspresija pojačava tijekom infekcije uglavnom su geni uključeni u upalne i imunološke procese, međutim neki pripadaju i skupini transkripcijskih faktora te genima povezanim s razvojem i diferencijacijom. Ovi rezultati u skladu su s dosadašnjim saznanjima o CMV-u kao virusu koji izaziva upalu te uzrokuje razvojne poremećaje tijekom kongenitalnih infekcija. Brojni geni čija se ekspresija smanjila tijekom infekcije povezani su sa funkcijama čija je uloga u infekciji za sada nepoznata poput dugačkih intergenskih nekodirajućih RNK, antisense RNK ili malih nukleolarnih RNK. GO analiza rezultirala je detekcijom disreguliranih bioloških puteva koji još do sada nisu bili povezani sa citomegalovirusnom infekcijom, a koji imaju potencijal rasvjetljavanja nekih nepoznanica u patogenezi citomegalovirusne infekcije. Zaključci Jedno od najznačajnijih otkrića u ovome radu jest dokaz izuzetne kompleksnosti transkriptoma MCMV-a koji dosad nije bio ovako sustavno istraživan niti su postojale transkriptomske mape. Ova analiza transkriptoma MCMV-a predstavlja važan prvi korak ka razvoju boljih genomskih mapa i reanotaciji genoma MCMV-a. Analiza odgovora stanica domaćina na infekciju dala je novi pogled na molekularne interakcije između virusa i domaćina i otvorila brojna nova područja istraživanja koja imaju potencijal da p pronađu nove mogućnosti liječenja bolesti izazvanih CMV-om. Ključne riječi mišji citomegalovirus, MCMV, transkriptom, ekspresija gena, izbjegavanje imunološko

Analiza transkriptoma mišijeg citomegalovirusa

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.Svrha istraživanja Humani citomegalovirus široko je rasprostranjen patogen, a posebno je opasan za trudnice, novorođenčad i imunosuprimirane pacijente. Nažalost, efikasnog cjepivo nema, a postoji potreba i za efikasnijim i manje toksičnim antiviralnim lijekovima. Glavne prepreke razvoju novih antiviralnih ljekova i cjepiva jesu: (1) specifičnost za vrstu i (2) praznine u našem znanju i razumjevanju virusnih gena, interakcijama virusnih gena i domaćina te kako te interakcije izazivaju bolest. Prva prepreka uspješno se nadvladava korištenjem animalnih virusa, posebice mišjeg citomegalovirusa (MCMV). S ciljem nadvladavanja druge prepreke u ovom je radu provedena detaljna analiza transkriptoma mišjeg citomegalovirusa (MCMV) te analiza transkriptoma stanica domaćina tijekom litičke infekcije citomegalovirusom. Materijali i metode Transkriptom MCMV analiziran je na dva načina: klasičnom analizom cDNK knjižnice i sekvencioniranjem dobivenih klonova te analizom transkriptoma uz pomoć sekvencioniranja nove generacije (odnosno RNK-sekvencioniranjem, eng. RNASeq) koja omogućava paralelno praćenje i transkriptoma domaćina uz transkriptom virusa. Analiza transkriptoma domaćina rezultira vrlo dugačkim listama diferencijalno reguliranih gena iz kojih je teško izvući neko biološko značenje. Stoga su liste diferencijalno reguliranih gena domaćina podvrgnute analizi termina genske ontologije (eng. gene ontology analysis odnosno GO analiza) i analizom dereguliranih bioloških puteva. Transkripcijski kompleksne regije genoma MCMV dodatno su analizirane Northern hibridizacijom i metodom RT-PCR dok je korelacija između količine transkripata i proteina odabranih gena analizirana metodom Western blot. Na kraju, funkcija novog, prekrojenog transkripta MAT (most abundant transcript; najzastupljeniji transkript) analizirana je uz pomoć reporterskih stanica koje nose aktivacijske Ly49 receptore. Rezultati Ovaj rad predstavlja prvu detaljnu analizu transkriptoma MCMV-a korištenjem komplementarnih metoda analize cDNK knjižnice i RNASeq analizom, a rezultirala je identifikacijom brojnih novih transkripata MCMV, uključujući i nove prekrojene transkripte kao i transkripte koji se prepisuju sa intergenskih regija. Ustanovljeno je da najjače izraženi virusni transkripti imaju nepoznatu funkciju i često su pogrešno anotirani. Najjače izražen transkript (tzv. MAT transkript) prvi je virusni transkript koji ima i kodirajuću i nekodirajuću funkciju. Naime, nedavno je pokazano da se na njegovom 3' netranslatiranom kraju (3' UTR, od eng. 3' untranslated region) nalazi vezno mjesto za staničnu mikro-RNK [18, 84], dok je u ovom radu pokazano da on također kodira i barem još dva proteina. Uz ove dvije navedene funkcije, u ovom je radu otkriveno da je 5' netranslatirani kraj (5'UTR) MAT transkripta bitan virusni faktor kojeg na inificranim stanicama prepoznaju stanice prirodne ubojice pomoću aktivacijskih receptora Ly49. Analiza transkriptoma stanica domaćina pokazala je da litička infekcija virusom MCMV izaziva izrazite promjene u ekspresijskom profilu gena domaćina: ekspresija gotovo trećine gena miša promijenila se uslijed infekcije virusom MCMV. Geni čija se ekspresija pojačava tijekom infekcije uglavnom su geni uključeni u upalne i imunološke procese, međutim neki pripadaju i skupini transkripcijskih faktora te genima povezanim s razvojem i diferencijacijom. Ovi rezultati u skladu su s dosadašnjim saznanjima o CMV-u kao virusu koji izaziva upalu te uzrokuje razvojne poremećaje tijekom kongenitalnih infekcija. Brojni geni čija se ekspresija smanjila tijekom infekcije povezani su sa funkcijama čija je uloga u infekciji za sada nepoznata poput dugačkih intergenskih nekodirajućih RNK, antisense RNK ili malih nukleolarnih RNK. GO analiza rezultirala je detekcijom disreguliranih bioloških puteva koji još do sada nisu bili povezani sa citomegalovirusnom infekcijom, a koji imaju potencijal rasvjetljavanja nekih nepoznanica u patogenezi citomegalovirusne infekcije. Zaključci Jedno od najznačajnijih otkrića u ovome radu jest dokaz izuzetne kompleksnosti transkriptoma MCMV-a koji dosad nije bio ovako sustavno istraživan niti su postojale transkriptomske mape. Ova analiza transkriptoma MCMV-a predstavlja važan prvi korak ka razvoju boljih genomskih mapa i reanotaciji genoma MCMV-a. Analiza odgovora stanica domaćina na infekciju dala je novi pogled na molekularne interakcije između virusa i domaćina i otvorila brojna nova područja istraživanja koja imaju potencijal da p pronađu nove mogućnosti liječenja bolesti izazvanih CMV-om. Ključne riječi mišji citomegalovirus, MCMV, transkriptom, ekspresija gena, izbjegavanje imunološko

Murine CMV Expressing the High Affinity NKG2D Ligand MULT-1: A Model for the Development of Cytomegalovirus-Based Vaccines

The development of a vaccine against human cytomegalovirus (CMV) has been a subject of long-term medical interest. The research during recent years identified CMV as an attractive vaccine vector against infectious diseases and tumors. The immune response to CMV persists over a lifetime and its unique feature is the inflationary T cell response to certain viral epitopes. CMV encodes numerous genes involved in immunoevasion, which are non-essential for virus growth in vitro. The deletion of those genes results in virus attenuation in vivo, which enables us to dramatically manipulate its virulence and the immune response. We have previously shown that the murine CMV (MCMV) expressing RAE-1γ, one of the cellular ligands for the NKG2D receptor, is highly attenuated in vivo but retains the ability to induce a strong CD8+ T cell response. Here, we demonstrate that recombinant MCMV expressing high affinity NKG2D ligand murine UL16 binding protein-like transcript (MULT-1) (MULT-1MCMV) inserted in the place of its viral inhibitor is dramatically attenuated in vivo in a NK cell-dependent manner, both in immunocompetent adult mice and in immunologically immature newborns. MULT-1MCMV was more attenuated than the recombinant virus expressing RAE-1γ. Despite the drastic sensitivity to innate immune control, MULT-1MCMV induced an efficient CD8+ T cell response to viral and vectored antigens. By using in vitro assay, we showed that similar to RAE-1γMCMV, MULT-1 expressing virus provided strong priming of CD8+ T cells. Moreover, MULT-1MCMV was able to induce anti-viral antibodies, which after passing the transplacental barrier protect offspring of immunized mothers from challenge infection. Altogether, this study further supports the concept that CMV expressing NKG2D ligand possesses excellent characteristics to serve as a vaccine or vaccine vector