97 research outputs found
Center for antibody production rijeka (capri)- towards a new resource for antibody-based tools
Center for Antibody Production Rijeka (CAPRI) is a part of the Center for Proteomics devoted to
mouse monoclonal antibody production. The Center for Proteomics is a department at the University of Rijeka
Faculty of Medicine that is to a large extent self-sustainable since it employs scientists, administrative and
technical staff and covers all its research costs from competitive international grants which makes it a unique
department in the Croatian academia. One of the objectives of the Center for Proteomics is to put emphasis on
applied R&D and utilization of the existing large collection of antibodies and antibody-based products, both
suitable for further commercial development
MCMV avoidance of recognition and control by NK cells
Natural killer (NK) cells play an important role in virus control during infection. Many viruses have developed mechanisms for subversion of NK cell responses. Murine cytomegalovirus (MCMV) is exceptionally successful in avoiding NK cell control. Here, we summarize the major MCMV evasion mechanisms targeting NK cell functions and their role in viral pathogenesis. The mechanisms by which NK cells regulate CD8+ T cell response, particularly with respect to the role of NK cell receptors recognizing viral antigens, are discussed. In addition, we discuss the role of NK cell receptors in generation and maintenance of memory NK cells. Final part of this review illustrates how the NK cell response and its viral regulation can be exploited in designing recombinant viral vectors able to induce robust and protective CD8+ T cell response
The interaction between CD300a and phosphatidylserine inhibits tumor cell killing by NK cells
The activity of NK cells is controlled by inhibitory and activating receptors. The inhibitory receptors interact mostly with MHC class I proteins, however, inhibitory receptors such as CD300a, which bind to non‐MHC class I ligands, also exist. Recently, it was discovered that phosphatidylserine (PS) is a ligand for CD300a and that the interaction between PS expressed on apoptotic cells and CD300a inhibits the uptake of apoptotic cells by phagocytic cells. Whether PS can inhibit NK‐cell activity through CD300a is unknown. Here, we have generated specific antibodies directed against CD300a and we used these mAbs to demonstrate that various NK‐cell clones express different levels of CD300a. We further demonstrated that both CD300a and its highly homologous molecule CD300c bind to the tumor cells equally well and that they recognize PS and additional unknown ligand(s) expressed by tumor cells. Finally, we showed that blocking the PS–CD300a interaction resulted in increased NK‐cell killing of tumor cells. Collectively, we demonstrate a new tumor immune evasion mechanism that is mediated through the interaction between PS and CD300a and we suggest that CD300c, similarly to CD300a, also interacts with PS
UL36 Rescues Apoptosis Inhibition and In vivo Replication of a Chimeric MCMV Lacking the M36 Gene
Apoptosis is an important defensemechanismmounted by the immune systemto control
virus replication. Hence, cytomegaloviruses (CMV) evolved and acquired numerous
anti-apoptotic genes. The product of the human CMV (HCMV) UL36 gene, pUL36 (also
known as vICA), binds to pro-caspase-8, thus inhibiting death-receptor apoptosis and
enabling viral replication in differentiated THP-1 cells. In vivo studies of the function of
HCMV genes are severely limited due to the strict host specificity of cytomegaloviruses,
but CMV orthologues that co-evolved with other species allow the experimental study
of CMV biology in vivo. The mouse CMV (MCMV) homolog of the UL36 gene is called
M36, and its protein product (pM36) is a functional homolog of vICA that binds to
murine caspase-8 and inhibits its activation. M36-deficient MCMV is severely growth
impaired in macrophages and in vivo. Here we show that pUL36 binds to the murine
pro-caspase-8, and that UL36 expression inhibits death-receptor apoptosis in murine
cells and can replace M36 to allow MCMV growth in vitro and in vivo. We generated a
chimeric MCMV expressing the UL36 ORF sequence instead of the M36 one. The newly
generatedMCMVUL36 inhibited apoptosis inmacrophage lines RAW264.7, J774A.1, and
IC-21 and its growth was rescued to wild type levels. Similarly, growth was rescued in vivo
in the liver and spleen, but only partially in the salivary glands of BALB/c and C57BL/6
mice. In conclusion, we determined that an immune-evasive HCMV gene is conserved
enough to functionally replace its MCMV counterpart and thus allow its study in an in vivo
setting. As UL36 and M36 proteins engage the same molecular host target, our newly
developed model can facilitate studies of anti-viral compounds targeting pUL36 in vivo
The herpesviral Fc receptor fcr-1 down-regulates the NKG2D ligands MULT-1 and H60
Members of the α- and β-subfamily of herpesviridae encode glycoproteins that specifically bind to the Fc part of immunoglobulin (Ig)G. Plasma membrane resident herpesviral Fc receptors seem to prevent virus-specific IgG from activating antibody-dependent effector functions. We show that the mouse cytomegalovirus (MCMV) molecule fcr-1 promotes a rapid down-regulation of NKG2D ligands murine UL16-binding protein like transcript (MULT)-1 and H60 from the cell surface. Deletion of the m138/fcr-1 gene from the MCMV genome attenuates viral replication to natural killer (NK) cell response in an NKG2D-dependent manner in vivo. A distinct N-terminal module within the fcr-1 ectodomain in conjunction with the fcr-1 transmembrane domain was required to dispose MULT-1 to degradation in lysosomes. In contrast, down-modulation of H60 required the complete fcr-1 ectodomain, implying independent modes of fcr-1 interaction with the NKG2D ligands. The results establish a novel viral strategy for down-modulating NK cell responses and highlight the impressive diversity of Fc receptor functions
NK cell activation through the NKG2D ligand MULT-1 is selectively prevented by the glycoprotein encoded by mouse cytomegalovirus gene m145
The NK cell–activating receptor NKG2D interacts with three different cellular ligands, all of which are regulated by mouse cytomegalovirus (MCMV). We set out to define the viral gene product regulating murine UL16-binding protein-like transcript (MULT)-1, a newly described NKG2D ligand. We show that MCMV infection strongly induces MULT-1 gene expression, but surface expression of this glycoprotein is nevertheless completely abolished by the virus. Screening a panel of MCMV deletion mutants defined the gene m145 as the viral regulator of MULT-1. The MCMV m145-encoded glycoprotein turned out to be necessary and sufficient to regulate MULT-1 by preventing plasma membrane residence of MULT-1. The importance of MULT-1 in NK cell regulation in vivo was confirmed by the attenuating effect of the m145 deletion that was lifted after NK cell depletion. Our findings underline the significance of escaping MULT-1/NKG2D signaling for viral survival and maintenance
Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97
Abstract: The nuclear phase of herpesvirus replication is regulated through the formation of
regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear
capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely
pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric
assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the
mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in
turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide
electron microscopy-based data demonstrating the association of both nuclear capsids and NEC
proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC
constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid
interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with
earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided
by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells,
nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with
pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking
and NEC-capsid interaction
Cytomegalovirus protein m154 perturbs the adaptor protein-1 compartment mediating broad-spectrum immune evasion
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
Varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency
Varicella-zoster virus (VZV) establishes lifelong neuronal latency in most humans world-wide, reactivating in one-third to cause herpes zoster and occasionally chronic pain. How VZV establishes, maintains and reactivates from latency is largely unknown. VZV transcription during latency is restricted to the latency-associated transcript (VLT) and RNA 63 (encoding ORF63) in naturally VZV-infected human trigeminal ganglia (TG). While significantly more abundant, VLT levels positively correlated with RNA 63 suggesting co-regulated transcription during latency. Here, we identify VLT-ORF63 fusion transcripts and confirm VLT-ORF63, but not RNA 63, expression in human TG neurons. During in vitro latency, VLT is transcribed, whereas VLT-ORF63 expression is induced by reactivation stimuli. One isoform of VLT-ORF63, encoding a fusion protein combining VLT and ORF63 proteins, induces broad viral gene transcription. Collectively, our findings show that VZV expresses a unique set of VLT-ORF63 transcripts, potentially involved in the transition from latency to lytic VZV infection
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
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