Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 29
Filter
Add more filters










Publication year range
1.
Carbohydr Polym ; 341: 122349, 2024 Oct 01.
Article in English | MEDLINE | ID: mdl-38876728

ABSTRACT

Meningococcal glycoconjugate vaccines sourced from capsular polysaccharides (CPSs) of pathogenic Neisseria meningitidis strains are well-established measures to prevent meningococcal disease. However, the exact structural factors responsible for antibody recognition are not known. CPSs of Neisseria meningitidis serogroups Y and W differ by a single stereochemical center, yet they evoke specific immune responses. Herein, we developed specific monoclonal antibodies (mAbs) targeting serogroups C, Y, and W and evaluated their ability to kill bacteria. We then used these mAbs to dissect structural elements responsible for carbohydrate-protein interactions. First, Men oligosaccharides were screened against the mAbs using ELISA to select putative lengths representing the minimal antigenic determinant. Next, molecular interaction features between the mAbs and serogroup-specific sugar fragments were elucidated using STD-NMR. Moreover, X-ray diffraction data with the anti-MenW CPS mAb enabled the elucidation of the sugar-antibody binding mode. Our findings revealed common traits in the epitopes of all three sialylated serogroups. The minimal binding epitopes typically comprise five to six repeating units. Moreover, the O-acetylation of the neuraminic acid moieties was fundamental for mAb binding. These insights hold promise for the rational design of optimized meningococcal oligosaccharides, opening new avenues for novel production methods, including chemical or enzymatic approaches.


Subject(s)
Antibodies, Monoclonal , Meningococcal Vaccines , Neisseria meningitidis , Polysaccharides, Bacterial , Serogroup , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/chemistry , Neisseria meningitidis/immunology , Neisseria meningitidis/chemistry , Meningococcal Vaccines/immunology , Meningococcal Vaccines/chemistry , Polysaccharides, Bacterial/immunology , Polysaccharides, Bacterial/chemistry , Antibodies, Bacterial/immunology , Epitopes/immunology , Epitopes/chemistry , Animals , Mice , Humans , Bacterial Capsules/immunology , Bacterial Capsules/chemistry , Antibody Formation/immunology
2.
Viruses ; 16(2)2024 01 30.
Article in English | MEDLINE | ID: mdl-38399983

ABSTRACT

Congenital human cytomegalovirus (HCMV) infection may cause life-threatening disease and permanent damage to the central nervous system. The mouse model of CMV infection is most commonly used to study mechanisms of infection and pathogenesis. While essential to limit mouse CMV (MCMV) replication, the inflammatory responses, particularly IFNγ and TNFα, cause neurodevelopmental abnormalities. Other soluble mediators of the immune response in most tissues remain largely unexplored. To address this gap, we quantified 48 soluble mediators of the immune response, including 32 cytokines, 10 chemokines, 3 growth factors/regulators, and 3 soluble receptors in the spleen, liver, lungs, and brain at 9 and 14 days postinfection (dpi). Our analysis found 25 induced molecules in the brain at 9 dpi, with an additional 8 showing statistically elevated responses at 14 dpi. Specifically, all analyzed CCL group cytokines (CCL2, CCL3, CCL4, CCL5, CCL7, and CCL11) were upregulated at 14 dpi in the brain. Furthermore, data revealed differentially regulated analytes across tissues, such as CCL11, CXCL5, and IL-10 in the brain, IL-33/IL-33R in the liver, and VEGF-a and IL-5 in the lungs. Overall, this study provides an overview of the immune dynamics of soluble mediators in congenital CMV.


Subject(s)
Cytomegalovirus Infections , Muromegalovirus , Animals , Humans , Mice , Cytokines , Brain , Tumor Necrosis Factor-alpha
3.
Pharmaceutics ; 15(12)2023 Nov 27.
Article in English | MEDLINE | ID: mdl-38140021

ABSTRACT

Despite the availability of currently approved antiviral drugs, infections with human cytomegalovirus (HCMV) still cause clinically challenging, sometimes life-threatening situations. There is an urgent need for enhanced anti-HCMV drugs that offer improved efficacy, reduced dosages and options for long-term treatment without risk of the development of viral drug resistance. Recently, we reported the pronounced anti-HCMV efficacy of pharmacological inhibitors of cyclin-dependent kinases (CDKs), in particular, the potential of utilizing drug synergies upon combination treatment with inhibitors of host CDKs and the viral CDK-like kinase pUL97 (vCDK/pUL97). Here, we expand this finding by further assessing the in vitro synergistic antiviral interaction between vCDK and CDK inhibitors towards HCMV as well as non-human cytomegaloviruses. An extension of this synergy approach was achieved in vivo by using the recombinant MCMV-UL97/mouse model, confirming the high potential of combination treatment with the clinically approved vCDK inhibitor maribavir (MBV) and the developmental CDK7 inhibitor LDC4297. Moreover, mechanistic aspects of this synergistic drug combination were illustrated on the levels of intracellular viral protein transport and viral genome replication. The analysis of viral drug resistance did not reveal resistance formation in the case of MBV + LDC4297 combination treatment. Spanning various investigational levels, these new results strongly support our concept, employing the great potential of anti-HCMV synergistic drug treatment.

4.
Animals (Basel) ; 13(11)2023 May 31.
Article in English | MEDLINE | ID: mdl-37889706

ABSTRACT

Bovine mastitis is the most frequent disease on dairy farms, which leads to a decrease in the health welfare of the animals and great economic losses. This study was aimed at determining the quantitative variations in the milk proteome caused by natural infection by Staphylococcus and Streptococcus species in order to gain further understanding of any discrepancies in pathophysiology and host immune responses, independent of the mastitis level. After identification of Staphylococcus (N = 51) and Streptococcus (N = 67) spp., tandem mass tag (TMT)-labeled quantitative proteomic and liquid chromatography-mass spectrometry (LC-MS/MS) techniques on a modular Ultimate 3000 RSLCnano system coupled to a Q Exactive Plus was applied on aseptically sampled milk from Holstein cows. Proteome Discoverer was used for protein identification and quantitation through the SEQUEST algorithm. Statistical analysis employing R was used to identify differentially abundant proteins between the groups. Protein classes, functions and functional-association networks were determined using the PANTHER and STRING tools and pathway over-representation using the REACTOME. In total, 156 master bovine proteins were identified (two unique peptides, p < 0.05 and FDR < 0.001), and 20 proteins showed significantly discrepant abundance between the genera (p < 0.05 and FDR < 0.5). The most discriminatory proteins per group were odorant-binding protein (higher in staphylococci) and fibrinogen beta chain protein (higher in streptococci). The receiver operating characteristic (ROC) curve showed that protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I have diagnostic potential for differentiating staphylococci and streptococci intramammary infection and inflammation. Improved understanding of the host response mechanisms and recognition of potential biomarkers of specific-pathogen mastitis, which may aid prompt diagnosis for control implementation, are potential benefits of this study.

5.
mBio ; 13(5): e0100722, 2022 10 26.
Article in English | MEDLINE | ID: mdl-36066102

ABSTRACT

Morphogenesis of herpesvirus particles is highly conserved; however, the capsid assembly and genome packaging of human cytomegalovirus (HCMV) exhibit unique features. Examples of these include the essential role of the small capsid protein (SCP) and the existence of the ß-herpesvirus-specific capsid-associated protein pp150. SCP and pp150, as well as the UL77 and UL93 proteins, are important capsid constituents, yet their precise mechanism of action is elusive. Here, we analyzed how deletion of the open reading frames (ORFs) encoding pUL77, pUL93, pp150, or SCP affects the protein composition of nuclear capsids. This was achieved by generating HCMV genomes lacking the respective genes, combined with a highly efficient transfection technique that allowed us to directly analyze these mutants in transfected cells. While no obvious effects were observed when pUL77, pUL93, or pp150 was missing, the absence of SCP impeded capsid assembly due to strongly reduced amounts of major capsid protein (MCP). Vice versa, when MCP was lacking, SCP became undetectable, indicating a mutual dependence of SCP and MCP for establishing appropriate protein levels. The SCP domain mediating stable MCP levels could be narrowed down to a C-terminal helix known to convey MCP binding. Interestingly, an SCP-EGFP (enhanced green fluorescent protein) fusion protein which also impaired the production of infectious progeny acted in a different manner, as capsid assembly was not abolished; however, SCP-EGFP-harboring capsids were devoid of DNA and trapped in paracrystalline nuclear structures. These results indicate that SCP is essential in HCMV because of its impact on MCP levels and reveal SCP as a potential target for antiviral inhibitors. IMPORTANCE Human cytomegalovirus (HCMV) is a ubiquitous pathogen causing life-threatening disease in immunocompromised individuals. Virus-specific processes such as capsid assembly and genome packaging can be exploited to design new antiviral strategies. Here, we report on a novel function of the HCMV small capsid protein (SCP), namely, ensuring stable levels of major capsid protein (MCP), thereby governing capsid assembly. Furthermore, we discovered a mutual dependence of the small and major capsid proteins to guarantee appropriate levels of the other respective protein and were able to pin down the SCP domain responsible for this effect to a region previously shown to mediate binding to the major capsid protein. In summary, our data contribute to the understanding of how SCP plays an essential part in the HCMV infection cycle. Moreover, disrupting the SCP-MCP interface may provide a starting point for the development of novel antiviral drugs.


Subject(s)
Capsid Proteins , Capsid , Humans , Capsid/metabolism , Capsid Proteins/metabolism , Cell Nucleus/metabolism , Cytomegalovirus/genetics , Cytomegalovirus/metabolism
6.
Viruses ; 14(2)2022 02 21.
Article in English | MEDLINE | ID: mdl-35216036

ABSTRACT

In early 2020, the COVID-19 pandemic sparked a global crisis that continues to pose a serious threat to human health and the economy. Further advancement in research is necessary and requires the availability of quality molecular tools, including monoclonal antibodies. Here, we present the development and characterization of a collection of over 40 new monoclonal antibodies directed against different SARS-CoV-2 proteins. Recombinant SARS-CoV-2 proteins were expressed, purified, and used as immunogens. Upon development of specific hybridomas, the obtained monoclonal antibody (mAb) clones were tested for binding to recombinant proteins and infected cells. We generated mAbs against structural proteins, the Spike and Nucleocapsid protein, several non-structural proteins (nsp1, nsp7, nsp8, nsp9, nsp10, nsp16) and accessory factors (ORF3a, ORF9b) applicable in flow cytometry, immunofluorescence, or Western blot. Our collection of mAbs provides a set of novel, highly specific tools that will allow a comprehensive analysis of the viral proteome, which will allow further understanding of SARS-CoV-2 pathogenesis and the design of therapeutic strategies.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Viral/pharmacology , SARS-CoV-2/immunology , Viral Proteins/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Antibodies, Monoclonal/classification , Antibodies, Viral/immunology , COVID-19/therapy , COVID-19/virology , HEK293 Cells , Humans , Recombinant Proteins/immunology , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/immunology
7.
J Biol Chem ; 298(3): 101625, 2022 03.
Article in English | MEDLINE | ID: mdl-35074430

ABSTRACT

Varicella-zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. The VZV Orf24-Orf27 complex represents the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled virus capsids from the nucleus. While previous studies have primarily emphasized that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focuses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. Here, we describe the crystal structure of the Orf24-Orf27 complex at 2.1 Å resolution. Coimmunoprecipitation and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of thermodynamic parameters of NEC formation of three prototypical α-, ß-, and γ herpesviruses, i.e., VZV, human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV), revealed highly similar binding affinities for the autologous interaction with specific differences in enthalpy and entropy. Computational alanine scanning, structural comparisons, and mutational data highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in ß- and γ-herpesviruses, including a salt bridge formed between Orf24-Arg167 and Orf27-Asp126. This interaction is located outside of the hook-into-groove interface and contributes significantly to the free energy of complex formation. Combined, these data explain distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings will prove valuable in attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates.


Subject(s)
Herpesvirus 3, Human , Nuclear Envelope , Viral Proteins , Crystallography, X-Ray , Herpesvirus 3, Human/chemistry , Herpesvirus 3, Human/genetics , Humans , Nuclear Envelope/chemistry , Nuclear Envelope/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Virus Release
8.
J Virol ; 96(2): e0087621, 2022 01 26.
Article in English | MEDLINE | ID: mdl-34705561

ABSTRACT

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 M116, 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 in vivo. 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. IMPORTANCE 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 M116 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 in vivo. 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.


Subject(s)
Mononuclear Phagocyte System/virology , Muromegalovirus/physiology , Viral Envelope Proteins/metabolism , Animals , Fibroblasts/metabolism , Fibroblasts/virology , Glycosylation , Herpesviridae Infections/virology , Membrane Glycoproteins/metabolism , Mice , Mononuclear Phagocyte System/metabolism , Transcription, Genetic , Viral Envelope Proteins/genetics , Virion/metabolism , Virus Assembly , Virus Internalization , Virus Replication
9.
PLoS Pathog ; 17(12): e1010175, 2021 12.
Article in English | MEDLINE | ID: mdl-34929007

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.


Subject(s)
Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/metabolism , Protein Domains , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , Binding Sites , Binding Sites, Antibody , COVID-19/prevention & control , Chlorocebus aethiops , Female , HEK293 Cells , Humans , Immunoglobulin Fc Fragments/therapeutic use , Immunoglobulin G/therapeutic use , Mice, Transgenic , Neutralization Tests , Protein Binding , Recombinant Fusion Proteins/therapeutic use , SARS-CoV-2/drug effects , Vero Cells
10.
Nat Commun ; 11(1): 6324, 2020 12 10.
Article in English | MEDLINE | ID: mdl-33303747

ABSTRACT

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.


Subject(s)
Gene Expression Regulation, Viral , Herpesvirus 3, Human/genetics , Sensory Receptor Cells/virology , Viral Proteins/genetics , Virus Activation/genetics , Virus Latency/genetics , Anisomycin/pharmacology , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Open Reading Frames/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, Genetic/drug effects , Trigeminal Ganglion/pathology , Trigeminal Ganglion/virology , Viral Proteins/metabolism
11.
Front Microbiol ; 11: 1179, 2020.
Article in English | MEDLINE | ID: mdl-32547533

ABSTRACT

Herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) are two closely related human alphaherpesviruses that persistently infect most adults worldwide and cause a variety of clinically important diseases. Herpesviruses are extremely well adapted to their hosts and interact broadly with cellular proteins to regulate virus replication and spread. However, it is incompletely understood how HSV-1 and VZV interact with the host proteome during productive infection. This study determined the temporal changes in virus and host protein expression during productive HSV-1 and VZV infection in the same cell type. Results demonstrated the temporally coordinated expression of HSV-1 and VZV proteins in infected cells. Analysis of the host proteomes showed that both viruses affected extracellular matrix composition, transcription, RNA processing and cell division. Moreover, the prominent role of epidermal growth factor receptor (EGFR) signaling during productive HSV-1 and VZV infection was identified. Stimulation and inhibition of EGFR leads to increased and decreased virus replication, respectively. Collectively, the comparative temporal analysis of viral and host proteomes in productively HSV-1 and VZV-infected cells provides a valuable resource for future studies aimed to identify target(s) for antiviral therapy development.

12.
Elife ; 92020 01 13.
Article in English | MEDLINE | ID: mdl-31928630

ABSTRACT

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.


Subject(s)
Adaptor Protein Complex 1/immunology , Immune Evasion/immunology , Membrane Proteins/metabolism , Muromegalovirus/physiology , Viral Proteins/metabolism , Animals , Cell Line , Down-Regulation , Humans , Membrane Proteins/genetics , Mice, Inbred BALB C , Mice, Inbred C57BL , Muromegalovirus/genetics , Viral Proteins/genetics
13.
Infect Immun ; 87(9)2019 09.
Article in English | MEDLINE | ID: mdl-31285252

ABSTRACT

Multidrug-resistant enterococci are major causes of hospital-acquired infections. Immunotherapy with monoclonal antibodies (MAbs) targeting bacterial antigens would be a valuable treatment option in this setting. Here, we describe the development of two MAbs through hybridoma technology that target antigens from the most clinically relevant enterococcal species. Diheteroglycan (DHG), a well-characterized capsular polysaccharide of Enterococcus faecalis, and the secreted antigen A (SagA), an immunogenic protein from Enterococcus faecium, are both immunogens that have been proven to raise opsonic and cross-reactive antibodies against enterococcal strains. For this purpose, a conjugated form of the native DHG with SagA was used to raise the antibodies in mice, while enzyme-linked immunosorbent assay and opsonophagocytic assay were combined in the selection process of hybridoma cells producing immunoreactive and opsonic antibodies targeting the selected antigens. From this process, two highly specific IgG1(κ) MAbs were obtained, one against the polysaccharide (DHG.01) and one against the protein (SagA.01). Both MAbs exhibited good opsonic killing against the target bacterial strains: DHG.01 showed 90% killing against E. faecalis type 2, and SagA.01 showed 40% killing against E. faecium 11231/6. In addition, both MAbs showed cross-reactivity toward other E. faecalis and E. faecium strains. The sequences from the variable regions of the heavy and light chains were reconstructed in expression vectors, and the activity of the MAbs upon expression in eukaryotic cells was confirmed with the same immunological assays. In summary, we identified two opsonic MAbs against enterococci which could be used for therapeutic or prophylactic approaches against enterococcal infections.


Subject(s)
Antibodies, Monoclonal/immunology , Drug Resistance, Microbial , Enterococcus faecalis/immunology , Enterococcus faecium/immunology , Immunotherapy/methods , Opsonin Proteins/immunology , Animals , Antigens, Bacterial/immunology , Bacterial Capsules/chemistry , Mice , Polysaccharides/immunology
14.
Cell Mol Immunol ; 16(1): 40-52, 2019 01.
Article in English | MEDLINE | ID: mdl-30275538

ABSTRACT

Poliovirus receptor (PVR, CD155) has recently been gaining scientific interest as a therapeutic target in the field of tumor immunology due to its prominent endogenous and immune functions. In contrast to healthy tissues, PVR is expressed at high levels in several human malignancies and seems to have protumorigenic and therapeutically attractive properties that are currently being investigated in the field of recombinant oncolytic virotherapy. More intriguingly, PVR participates in a considerable number of immunoregulatory functions through its interactions with activating and inhibitory immune cell receptors. These functions are often modified in the tumor microenvironment, contributing to tumor immunosuppression. Indeed, increasing evidence supports the rationale for developing strategies targeting these interactions, either in terms of checkpoint therapy (i.e., targeting inhibitory receptors) or in adoptive cell therapy, which targets PVR as a tumor marker.


Subject(s)
Neoplasms/therapy , Receptors, Immunologic/metabolism , Receptors, Virus/metabolism , Animals , Clinical Trials as Topic , Humans , Immunotherapy, Adoptive , Protein Binding , Receptors, Virus/chemistry
15.
J Proteome Res ; 17(3): 1269-1277, 2018 03 02.
Article in English | MEDLINE | ID: mdl-29441788

ABSTRACT

Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypoglycosylated forms were expressed in CHO, CHO-Lec1, and HEK293T cells. Fully nonglycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin. Wild-type and hypo/nonglycosylated rhAFM were purified from cell culture supernatants by immobilized metal ion affinity and size exclusion chromatography. Glycan analysis of purified proteins demonstrated differences in micro- and macro-heterogeneity of glycosylation enabling the comparison between hypoglycosylated, wild-type rhAFM, and native plasma afamin. Because antibody fragments can work as artificial chaperones by stabilizing the structure of proteins and consequently enhance the chance for successful crystallization, we incubated a Fab fragment of the monoclonal anti-afamin antibody N14 with human afamin and obtained a stoichiometric complex. Subsequent results showed sufficient expression of various partially or nonglycosylated forms of rhAFM in HEK293T and CHO cells and revealed that glycosylation is not necessary for expression and secretion.


Subject(s)
Antibodies, Monoclonal/chemistry , Antigen-Antibody Complex/chemistry , Carrier Proteins/chemistry , Glycoproteins/chemistry , Immunoglobulin Fab Fragments/chemistry , Protein Processing, Post-Translational , Serum Albumin, Human/chemistry , Amino Acid Sequence , Animals , Antibodies, Monoclonal/metabolism , Antigen-Antibody Complex/metabolism , CHO Cells , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cloning, Molecular , Cricetulus , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Glycoproteins/genetics , Glycoproteins/metabolism , Glycosylation , HEK293 Cells , Humans , Immunoglobulin Fab Fragments/metabolism , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/chemistry , Polysaccharides/chemistry , Polysaccharides/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serum Albumin, Human/genetics , Serum Albumin, Human/metabolism
16.
Viruses ; 10(1)2018 01 13.
Article in English | MEDLINE | ID: mdl-29342872

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.


Subject(s)
Capsid/physiology , Cytomegalovirus/enzymology , Cytomegalovirus/physiology , Nuclear Envelope/virology , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Kinases/metabolism , Cell Nucleus/ultrastructure , Cell Nucleus/virology , Cytomegalovirus/ultrastructure , Host-Pathogen Interactions , Humans , Immunohistochemistry , Microscopy, Electron , Nuclear Envelope/ultrastructure , Nuclear Lamina/ultrastructure , Nuclear Lamina/virology , Phosphorylation , Viral Proteins/metabolism , Virus Assembly , Virus Release , Virus Replication
17.
Article in English | MEDLINE | ID: mdl-28770171

ABSTRACT

Apoptosis is an important defense mechanism mounted by the immune system to 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 generated MCMVUL36 inhibited apoptosis in macrophage lines RAW 264.7, 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.


Subject(s)
Apoptosis , Host-Pathogen Interactions , Muromegalovirus/immunology , Muromegalovirus/physiology , Viral Proteins/metabolism , Virus Replication , Animals , Cell Line , Cytomegalovirus/genetics , Genetic Complementation Test , Herpesviridae Infections/pathology , Herpesviridae Infections/virology , Humans , Mice, Inbred BALB C , Mice, Inbred C57BL , Muromegalovirus/genetics , Viral Proteins/genetics
18.
J Virol ; 91(20)2017 10 15.
Article in English | MEDLINE | ID: mdl-28747504

ABSTRACT

The neurotropic herpesvirus varicella-zoster virus (VZV) establishes a lifelong latent infection in humans following primary infection. The low abundance of VZV nucleic acids in human neurons has hindered an understanding of the mechanisms that regulate viral gene transcription during latency. To overcome this critical barrier, we optimized a targeted capture protocol to enrich VZV DNA and cDNA prior to whole-genome/transcriptome sequence analysis. Since the VZV genome is remarkably stable, it was surprising to detect that VZV32, a VZV laboratory strain with no discernible growth defect in tissue culture, contained a 2,158-bp deletion in open reading frame (ORF) 12. Consequently, ORF 12 and 13 protein expression was abolished and Akt phosphorylation was inhibited. The discovery of the ORF 12 deletion, revealed through targeted genome sequencing analysis, points to the need to authenticate the VZV genome when the virus is propagated in tissue culture.IMPORTANCE Viruses isolated from clinical samples often undergo genetic modifications when cultured in the laboratory. Historically, VZV is among the most genetically stable herpesviruses, a notion supported by more than 60 complete genome sequences from multiple isolates and following multiple in vitro passages. However, application of enrichment protocols to targeted genome sequencing revealed the unexpected deletion of a significant portion of VZV ORF 12 following propagation in cultured human fibroblast cells. While the enrichment protocol did not introduce bias in either the virus genome or transcriptome, the findings indicate the need for authentication of VZV by sequencing when the virus is propagated in tissue culture.


Subject(s)
DNA, Viral/isolation & purification , Genome, Viral , Herpesvirus 3, Human/genetics , Open Reading Frames , Sequence Deletion , Cell Line , DNA, Complementary , Herpesvirus 3, Human/growth & development , Humans , Sequence Analysis, DNA/methods , Transcriptome , Viral Proteins , Virion , Virus Latency
19.
PLoS Pathog ; 13(5): e1006346, 2017 May.
Article in English | MEDLINE | ID: mdl-28542541

ABSTRACT

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans.


Subject(s)
Chickenpox/virology , Herpes Zoster/virology , Herpesvirus 3, Human/genetics , Leukocytes/physiology , Viral Envelope Proteins/genetics , Animals , Cell Line , Cell Movement , Chemokines/metabolism , Chickenpox/immunology , Drosophila melanogaster , Epithelial Cells/virology , Genes, Reporter , Herpes Zoster/immunology , Herpesvirus 3, Human/immunology , Herpesvirus 3, Human/physiology , Host-Pathogen Interactions , Humans , Mutation , Palatine Tonsil/virology , Protein Domains , T-Lymphocytes/virology , Viral Envelope Proteins/immunology , Viral Envelope Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Virion
20.
Acta Crystallogr D Struct Biol ; 72(Pt 12): 1267-1280, 2016 12 01.
Article in English | MEDLINE | ID: mdl-27917827

ABSTRACT

The monoclonal antibody N14 is used as a detection antibody in ELISA kits for the human glycoprotein afamin, a member of the albumin family, which has recently gained interest in the capture and stabilization of Wnt signalling proteins, and for its role in metabolic syndrome and papillary thyroid carcinoma. As a rare occurrence, the N14 Fab is N-glycosylated at Asn26L at the onset of the VL1 antigen-binding loop, with the α-1-6 core fucosylated complex glycan facing out of the L1 complementarity-determining region. The crystal structures of two non-apparent (pseudo) isomorphous crystals of the N14 Fab were analyzed, which differ significantly in the elbow angles, thereby cautioning against the overinterpretation of domain movements upon antigen binding. In addition, the map quality at 1.9 Šresolution was sufficient to crystallographically re-sequence the variable VL and VH domains and to detect discrepancies in the hybridoma-derived sequence. Finally, a conservatively refined parsimonious model is presented and its statistics are compared with those from a less conservatively built model that has been modelled more enthusiastically. Improvements to the PDB validation reports affecting ligands, clashscore and buried surface calculations are suggested.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Carrier Proteins/immunology , Glycoproteins/immunology , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/immunology , Serum Albumin/immunology , Animals , Antigen-Antibody Complex , Complementarity Determining Regions , Crystallography, X-Ray , Glycosylation , Humans , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/immunology , Mice , Models, Molecular , Serum Albumin, Human
SELECTION OF CITATIONS
SEARCH DETAIL
...