Publications by authors named "Daniëlle Horst"

19 Publications

  • Page 1 of 1

Patient-Reported Experiences with a Relicensed Generic: Thioguanine for the Treatment of Inflammatory Bowel Diseases.

J Gastrointestin Liver Dis 2018 Dec;27(4):385-389

Department of Gastroenterology and Hepatology, Amsterdam UMC, Vrije Universiteit Amsterdam, AG and M Research Institute, Amsterdam, The Netherlands.

Background And Aim: Patient-reported outcomes and experiences are indicative of the impact and the quality of care. Thioguanine, a generic drug initially developed for leukemia, has been explored and relicensed as a certified treatment for patients with inflammatory bowel diseases (IBD). The patients' perception of this treatment has not been evaluated before. In this study, we aimed to assess self-reported experiences with thioguanine for IBD.

Methods: Questionnaires were sent out to members of the Dutch National Crohn's and Colitis patient organization. The Treatment Satisfaction with Medicines Questionnaire (SATMED-Q) was used to address questions regarding the satisfaction and impact of thioguanine therapy on the disease and their daily life. Furthermore, data on demographics, disease and (historical) treatment characteristics were collected. Open-ended questions were used for additional comments to the questionnaire.

Results: A total of 173 organization members (73% female) reported to be previous or current users of thioguanine. A total of 74% were satisfied with the effectiveness of thioguanine, whereas 5% were not. Eighty percent of the respondents were satisfied with the quality of care. A good or excellent impact on daily life was reported by 54%. A neutral or bad impact on daily life was reported by 40% and 6%, respectively. Improvement of disease activity was reported by 58%. This remained stable or worsened in 39% and 3%, respectively.

Conclusion: In this self-report survey, among thioguanine treated patients with IBD who had failed with traditional therapies, 80% reported satisfaction with medical care and 74% with the effectiveness of the therapy. In the evaluation of new or rediscovered therapies, patient-reported outcomes and experiences should be considered as a key instrument.
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http://dx.doi.org/10.15403/jgld.2014.1121.274.torDOI Listing
December 2018

Viral inhibition of the transporter associated with antigen processing (TAP): a striking example of functional convergent evolution.

PLoS Pathog 2015 Apr 16;11(4):e1004743. Epub 2015 Apr 16.

Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.

Herpesviruses are large DNA viruses that are highly abundant within their host populations. Even in the presence of a healthy immune system, these viruses manage to cause lifelong infections. This persistence is partially mediated by the virus entering latency, a phase of infection characterized by limited viral protein expression. Moreover, herpesviruses have devoted a significant part of their coding capacity to immune evasion strategies. It is believed that the close coexistence of herpesviruses and their hosts has resulted in the evolution of viral proteins that specifically attack multiple arms of the host immune system. Cytotoxic T lymphocytes (CTLs) play an important role in antiviral immunity. CTLs recognize their target through viral peptides presented in the context of MHC molecules at the cell surface. Every herpesvirus studied to date encodes multiple immune evasion molecules that effectively interfere with specific steps of the MHC class I antigen presentation pathway. The transporter associated with antigen processing (TAP) plays a key role in the loading of viral peptides onto MHC class I molecules. This is reflected by the numerous ways herpesviruses have developed to block TAP function. In this review, we describe the characteristics and mechanisms of action of all known virus-encoded TAP inhibitors. Orthologs of these proteins encoded by related viruses are identified, and the conservation of TAP inhibition is discussed. A phylogenetic analysis of members of the family Herpesviridae is included to study the origin of these molecules. In addition, we discuss the characteristics of the first TAP inhibitor identified outside the herpesvirus family, namely, in cowpox virus. The strategies of TAP inhibition employed by viruses are very distinct and are likely to have been acquired independently during evolution. These findings and the recent discovery of a non-herpesvirus TAP inhibitor represent a striking example of functional convergent evolution.
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http://dx.doi.org/10.1371/journal.ppat.1004743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4399834PMC
April 2015

Cowpox virus protein CPXV012 eludes CTLs by blocking ATP binding to TAP.

J Immunol 2014 Aug 14;193(4):1578-89. Epub 2014 Jul 14.

Department of Medical Microbiology, University Medical Center Utrecht, 3584 CX Utrecht, the Netherlands;

CD8(+) CTLs detect virus-infected cells through recognition of virus-derived peptides presented at the cell surface by MHC class I molecules. The cowpox virus protein CPXV012 deprives the endoplasmic reticulum (ER) lumen of peptides for loading onto newly synthesized MHC class I molecules by inhibiting the transporter associated with Ag processing (TAP). This evasion strategy allows the virus to avoid detection by the immune system. In this article, we show that CPXV012, a 9-kDa type II transmembrane protein, prevents peptide transport by inhibiting ATP binding to TAP. We identified a segment within the ER-luminal domain of CPXV012 that imposes the block in peptide transport by TAP. Biophysical studies show that this domain has a strong affinity for phospholipids that are also abundant in the ER membrane. We discuss these findings in an evolutionary context and show that a frameshift deletion in the CPXV012 gene in an ancestral cowpox virus created the current form of CPXV012 that is capable of inhibiting TAP. In conclusion, our findings indicate that the ER-luminal domain of CPXV012 inserts into the ER membrane, where it interacts with TAP. CPXV012 presumably induces a conformational arrest that precludes ATP binding to TAP and, thus, activity of TAP, thereby preventing the presentation of viral peptides to CTLs.
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http://dx.doi.org/10.4049/jimmunol.1400964DOI Listing
August 2014

Evaluation of viral interference with MHC class I-restricted antigen processing and presentation using a flow cytometry-based approach.

Methods Mol Biol 2013 ;960:127-136

Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.

The peptide content of MHC class I molecules present at the cell surface is monitored by surveilling CD8(+) cytotoxic T cells. In case of a viral infection, a proportion of the MHC class I molecules will carry peptides derived from viral proteins. This allows the CD8(+) T cells to recognize and eliminate virus-infected cells. This highly sensitive detection system of the host is counteracted by viruses, which have acquired functions to downregulate cell surface expression of MHC class I molecules. In this chapter, we describe a flow cytometry-based method to identify viral gene product(s) responsible for evasion from MHC class I-restricted antigen presentation. To this end, cells are transiently transfected using polyethylenimine (PEI) as a transfection reagent, followed by cell surface staining with MHC class I-specific monoclonal antibodies. Once viral proteins responsible for MHC class I downregulation have been identified, their mechanism of action can be characterized. Identification and characterization of virus-encoded MHC class I inhibitors augments our understanding of virus-host interactions and often provides new insights into antigen processing and presentation pathways, including related cellular processes such as protein trafficking and degradation.
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http://dx.doi.org/10.1007/978-1-62703-218-6_10DOI Listing
June 2013

Hiding lipid presentation: viral interference with CD1d-restricted invariant natural killer T (iNKT) cell activation.

Viruses 2012 Oct 23;4(10):2379-99. Epub 2012 Oct 23.

Department of Medical Microbiology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands.

The immune system plays a major role in protecting the host against viral infection. Rapid initial protection is conveyed by innate immune cells, while adaptive immunity (including T lymphocytes) requires several days to develop, yet provides high specificity and long-lasting memory. Invariant natural killer T (iNKT) cells are an unusual subset of T lymphocytes, expressing a semi-invariant T cell receptor together with markers of the innate NK cell lineage. Activated iNKT cells can exert direct cytolysis and can rapidly release a variety of immune-polarizing cytokines, thereby regulating the ensuing adaptive immune response. iNKT cells recognize lipids in the context of the antigen-presenting molecule CD1d. Intriguingly, CD1d-restricted iNKT cells appear to play a critical role in anti-viral defense: increased susceptibility to disseminated viral infections is observed both in patients with iNKT cell deficiency as well as in CD1d- and iNKT cell-deficient mice. Moreover, viruses have recently been found to use sophisticated strategies to withstand iNKT cell-mediated elimination. This review focuses on CD1d-restricted lipid presentation and the strategies viruses deploy to subvert this pathway.
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http://dx.doi.org/10.3390/v4102379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497057PMC
October 2012

Viral interference with antigen presentation: trapping TAP.

Mol Immunol 2013 Sep 8;55(2):139-42. Epub 2012 Nov 8.

Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.

Following primary infection, herpesviruses persist for life in their hosts, even when vigorous anti-viral immunity has been induced. Failure of the host immune system to eliminate infected cells is facilitated by highly effective immune evasion strategies acquired by these herpesviruses during millions of years of co-evolution with their hosts. Here, we review the mechanisms of action of viral gene products that lead to cytotoxic T cell evasion through interference with the function of the transporter associated with antigen processing, TAP. The viral TAP inhibitors impede transport of peptides from the cytosol into the ER lumen, thereby preventing peptide loading onto MHC class I complexes. Recent insights have revealed a pattern of functional convergent evolution. In every herpesvirus subfamily, inhibitors of TAP function have been identified that are, surprisingly, unrelated in genome location, structure, and mechanism of action. Recently, cowpox virus has also been found to encode a TAP inhibitor. Expanding our knowledge on how viruses perturb antigen presentation, in particular by targeting TAP, not only provides information on viral pathogenesis, but also reveals novel aspects of the cellular processes corrupted by these viruses, notably the translocation of peptides by the ATP-binding cassette (ABC) transporter TAP. As the various TAP inhibitors are anticipated to impede discrete conformational transitions it is expected that crystal structures of TAP-inhibitor complexes will reveal valuable structural information on the actual mechanism of peptide translocation by TAP. Viral TAP inhibitors are also used for various (clinical) applications, for example, as effective tools in antigen presentation studies and as immunomodulators in immunotherapy for cancer, heterologous vaccination, and transplant protection.
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http://dx.doi.org/10.1016/j.molimm.2012.10.009DOI Listing
September 2013

The "Bridge" in the Epstein-Barr virus alkaline exonuclease protein BGLF5 contributes to shutoff activity during productive infection.

J Virol 2012 Sep 13;86(17):9175-87. Epub 2012 Jun 13.

Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, Netherlands.

Replication of the human herpesvirus Epstein-Barr virus drastically impairs cellular protein synthesis. This shutoff phenotype results from mRNA degradation upon expression of the early lytic-phase protein BGLF5. Interestingly, BGLF5 is the viral DNase, or alkaline exonuclease, homologues of which are present throughout the herpesvirus family. During productive infection, this DNase is essential for processing and packaging of the viral genome. In contrast to this widely conserved DNase activity, shutoff is only mediated by the alkaline exonucleases of the subfamily of gammaherpesviruses. Here, we show that BGLF5 can degrade mRNAs of both cellular and viral origin, irrespective of polyadenylation. Furthermore, shutoff by BGLF5 induces nuclear relocalization of the cytosolic poly(A) binding protein. Guided by the recently resolved BGLF5 structure, mutants were generated and analyzed for functional consequences on DNase and shutoff activities. On the one hand, a point mutation destroying DNase activity also blocks RNase function, implying that both activities share a catalytic site. On the other hand, other mutations are more selective, having a more pronounced effect on either DNA degradation or shutoff. The latter results are indicative of an oligonucleotide-binding site that is partially shared by DNA and RNA. For this, the flexible "bridge" that crosses the active-site canyon of BGLF5 appears to contribute to the interaction with RNA substrates. These findings extend our understanding of the molecular basis for the shutoff function of BGLF5 that is conserved in gammaherpesviruses but not in alpha- and betaherpesviruses.
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http://dx.doi.org/10.1128/JVI.00309-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3416140PMC
September 2012

Epstein-Barr virus isolates retain their capacity to evade T cell immunity through BNLF2a despite extensive sequence variation.

J Virol 2012 Jan 19;86(1):572-7. Epub 2011 Oct 19.

Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.

The Epstein-Barr virus (EBV)-encoded immune evasion protein BNLF2a inhibits the transporter associated with antigen processing (TAP), thereby downregulating HLA class I expression at the cell surface. As a consequence, recognition of EBV-infected cells by cytotoxic T cells is impaired. Here, we show that sequence polymorphism of the BNLF2a protein is observed with natural EBV isolates, with evidence for positive selection. Despite these mutations, the BNLF2a variants efficiently reduce cell surface HLA class I levels. This conservation of BNLF2a function during evolution of EBV implies an important role for the viral TAP inhibitor in preventing T cell recognition during viral infection.
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http://dx.doi.org/10.1128/JVI.05151-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255881PMC
January 2012

Exploiting human herpesvirus immune evasion for therapeutic gain: potential and pitfalls.

Immunol Cell Biol 2011 Mar 8;89(3):359-66. Epub 2011 Feb 8.

Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.

Herpesviruses stand out for their capacity to establish lifelong infections of immunocompetent hosts, generally without causing overt symptoms. Herpesviruses are equipped with sophisticated immune evasion strategies, allowing these viruses to persist for life despite the presence of a strong antiviral immune response. Although viral evasion tactics appear to target virtually any stage of the innate and adaptive host immune response, detailed knowledge is now available on the molecular mechanisms underlying herpesvirus obstruction of MHC class I-restricted antigen presentation to T cells. This opens the way for clinical application. Here, we review and discuss recent efforts to exploit human herpesvirus MHC class I evasion strategies for the rational design of novel strategies for vaccine development, cancer treatment, transplant protection and gene therapy.
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http://dx.doi.org/10.1038/icb.2010.129DOI Listing
March 2011

EBV protein BNLF2a exploits host tail-anchored protein integration machinery to inhibit TAP.

J Immunol 2011 Mar 4;186(6):3594-605. Epub 2011 Feb 4.

Department of Medical Microbiology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands.

EBV, the prototypic human γ(1)-herpesvirus, persists for life in infected individuals, despite the presence of vigorous antiviral immunity. CTLs play an important role in the protection against viral infections, which they detect through recognition of virus-encoded peptides presented in the context of HLA class I molecules at the cell surface. The viral peptides are generated in the cytosol and are transported into the endoplasmic reticulum (ER) by TAP. The EBV-encoded lytic-phase protein BNLF2a acts as a powerful inhibitor of TAP. Consequently, loading of antigenic peptides onto HLA class I molecules is hampered, and recognition of BNLF2a-expressing cells by cytotoxic T cells is avoided. In this study, we characterize BNLF2a as a tail-anchored (TA) protein and elucidate its mode of action. Its hydrophilic N-terminal domain is located in the cytosol, whereas its hydrophobic C-terminal domain is inserted into membranes posttranslationally. TAP has no role in membrane insertion of BNLF2a. Instead, Asna1 (also named TRC40), a cellular protein involved in posttranslational membrane insertion of TA proteins, is responsible for integration of BNLF2a into the ER membrane. Asna1 is thereby required for efficient BNLF2a-mediated HLA class I downregulation. To optimally accomplish immune evasion, BNLF2a is composed of two specialized domains: its C-terminal tail anchor ensures membrane integration and ER retention, whereas its cytosolic N terminus accomplishes inhibition of TAP function. These results illustrate how EBV exploits a cellular pathway for TA protein biogenesis to achieve immune evasion, and they highlight the exquisite adaptation of this virus to its host.
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http://dx.doi.org/10.4049/jimmunol.1002656DOI Listing
March 2011

Viral evasion of T cell immunity: ancient mechanisms offering new applications.

Curr Opin Immunol 2011 Feb 9;23(1):96-103. Epub 2010 Dec 9.

Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.

Upon infecting a host, viruses are confronted by a coordinated and multi-faceted immune response. Indeed, evolutionary combat between virus and host has contributed signally to the host's development of a formidable innate and adaptive immune defense arsenal, and to the virus' acquisition of effective means to evade it. Cytotoxic T lymphocytes play a key role in the elimination of virus-infected cells, which they detect through recognition of virus-derived peptides displayed at the cell surface in the context of MHC class I molecules. This highly sensitive recognition system is a prime target for immune evasion strategies deployed by many viruses, particularly large DNA viruses such as herpesviruses and poxviruses. Elucidation of the mode of action of the immune evasion proteins encoded by these viruses has not only provided new insights into viral pathogenesis, but has also led to the discovery of hitherto unknown cell biological and immunological phenomena. Moreover, viral immune evasion proteins constitute extremely useful tools to block defined stages of the MHC class I presentation pathway, not only for research purposes, but also for clinical applications.
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http://dx.doi.org/10.1016/j.coi.2010.11.005DOI Listing
February 2011

Cowpox virus inhibits the transporter associated with antigen processing to evade T cell recognition.

Cell Host Microbe 2009 Nov;6(5):433-45

Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR 97006, USA.

Cowpox virus encodes an extensive array of putative immunomodulatory proteins, likely contributing to its wide host range, which includes zoonotic infections in humans. Unlike Vaccinia virus, cowpox virus prevents stimulation of CD8(+) T cells, a block that correlated with retention of MHC class I in the endoplasmic reticulum by the cowpox virus protein CPXV203. However, deletion of CPXV203 did not restore MHC class I transport or T cell stimulation. Here, we demonstrate the contribution of an additional viral protein, CPXV12, which interferes with MHC class I/peptide complex formation by inhibiting peptide translocation by the transporter associated with antigen processing (TAP). Importantly, human and mouse MHC class I transport and T cell stimulation was restored upon deletion of both CPXV12 and CPXV203, suggesting that these unrelated proteins independently mediate T cell evasion in multiple hosts. CPXV12 is a truncated version of a putative NK cell ligand, indicating that poxviral gene fragments can encode new, unexpected functions.
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http://dx.doi.org/10.1016/j.chom.2009.09.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2791678PMC
November 2009

Stage-specific inhibition of MHC class I presentation by the Epstein-Barr virus BNLF2a protein during virus lytic cycle.

PLoS Pathog 2009 Jun 26;5(6):e1000490. Epub 2009 Jun 26.

School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

The gamma-herpesvirus Epstein-Barr virus (EBV) persists for life in infected individuals despite the presence of a strong immune response. During the lytic cycle of EBV many viral proteins are expressed, potentially allowing virally infected cells to be recognized and eliminated by CD8+ T cells. We have recently identified an immune evasion protein encoded by EBV, BNLF2a, which is expressed in early phase lytic replication and inhibits peptide- and ATP-binding functions of the transporter associated with antigen processing. Ectopic expression of BNLF2a causes decreased surface MHC class I expression and inhibits the presentation of indicator antigens to CD8+ T cells. Here we sought to examine the influence of BNLF2a when expressed naturally during EBV lytic replication. We generated a BNLF2a-deleted recombinant EBV (DeltaBNLF2a) and compared the ability of DeltaBNLF2a and wild-type EBV-transformed B cell lines to be recognized by CD8+ T cell clones specific for EBV-encoded immediate early, early and late lytic antigens. Epitopes derived from immediate early and early expressed proteins were better recognized when presented by DeltaBNLF2a transformed cells compared to wild-type virus transformants. However, recognition of late antigens by CD8+ T cells remained equally poor when presented by both wild-type and DeltaBNLF2a cell targets. Analysis of BNLF2a and target protein expression kinetics showed that although BNLF2a is expressed during early phase replication, it is expressed at a time when there is an upregulation of immediate early proteins and initiation of early protein synthesis. Interestingly, BNLF2a protein expression was found to be lost by late lytic cycle yet DeltaBNLF2a-transformed cells in late stage replication downregulated surface MHC class I to a similar extent as wild-type EBV-transformed cells. These data show that BNLF2a-mediated expression is stage-specific, affecting presentation of immediate early and early proteins, and that other evasion mechanisms operate later in the lytic cycle.
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http://dx.doi.org/10.1371/journal.ppat.1000490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2695766PMC
June 2009

Reduced human leukocyte antigen expression in advanced-stage Ewing sarcoma: implications for immune recognition.

J Pathol 2009 Jun;218(2):222-31

Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Ewing sarcoma (EWS) is a tumour most commonly arising in bone, although on occasion in soft tissue, with a poor prognosis in patients with refractory or relapsed disease, despite multimodal therapy. Immunotherapeutic strategies based on tumour-reactive T and/or natural killer cells may improve the treatment of advanced-stage EWS. Since cellular immune recognition critically depends on human leukocyte antigen (HLA) expression, knowledge about HLA expression in EWS is crucial in the design of cellular immunotherapeutic strategies. Constitutive and IFNgamma-induced HLA class I expression was analysed in EWS cell lines (n = 6) by flow cytometry, using antibodies against both monomorphic and allele-specific antigens. Expression of antigen processing pathway components and beta-2 microglobulin (beta2m) was assessed by western blot. Expression of class II transactivator (CIITA), and its contribution to HLA class II expression, was evaluated by qRT-PCR, transduction assays, and flow cytometry. beta2m/HLA class I and class II expression was validated in EWS tumours (n = 67) by immunofluorescence. Complete or partial absence of HLA class I expression was observed in 79% of EWS tumours. Lung metastases consistently lacked HLA class I and sequential tumours demonstrated a tendency towards decreased expression upon disease progression. Together with absent or low constitutive expression levels of specific HLA class I loci and alleles, and differential induction of identical alleles by IFNgamma in different cell lines, these results may reflect the existence of an immune escape mechanism. Inducible expression of TAP-1/-2, tapasin, LMP-2/-7, and the beta2m/HLA class I complex by IFNgamma suggests that regulatory mechanisms are mainly responsible for heterogeneity in constitutive class I expression. EWSs lack IFNgamma-inducible HLA class II, due to lack of functional CIITA. The majority of EWS tumours, particularly if advanced-stage, exhibit complete or partial absence of both classes of HLA. This knowledge will be instrumental in the design of cellular immunotherapeutic strategies for advanced-stage EWS.
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http://dx.doi.org/10.1002/path.2537DOI Listing
June 2009

Specific targeting of the EBV lytic phase protein BNLF2a to the transporter associated with antigen processing results in impairment of HLA class I-restricted antigen presentation.

J Immunol 2009 Feb;182(4):2313-24

Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands.

EBV persists for life in the human host while facing vigorous antiviral responses that are induced upon primary infection. This persistence supports the idea that herpesviruses have acquired dedicated functions to avoid immune elimination. The recently identified EBV gene product BNLF2a blocks TAP. As a result, reduced amounts of peptides are transported by TAP from the cytoplasm into the endoplasmic reticulum (ER) lumen for binding to newly synthesized HLA class I molecules. Thus, BNLF2a perturbs detection by cytotoxic T cells. The 60-aa-long BNLF2a protein prevents the binding of both peptides and ATP to TAP, yet further mechanistic insight is, to date, lacking. In this study, we report that EBV BNLF2a represents a membrane-associated protein that colocalizes with its target TAP in subcellular compartments, primarily the ER. In cells devoid of TAP, expression levels of BNLF2a protein are greatly diminished, while ER localization of the remaining BNLF2a is retained. For interactions of BNLF2a with the HLA class I peptide-loading complex, the presence of TAP2 is essential, whereas tapasin is dispensible. Importantly, we now show that in B cells supporting EBV lytic replication, the BNLF2a protein is expressed early in infection, colocalizing and associating with the peptide-loading complex. These results imply that, during productive EBV infection, BNLF2a contributes to TAP inhibition and surface HLA class I down-regulation. In this way, EBV BNLF2a-mediated evasion from HLA class I-restricted T cell immunity contributes to creating a window for undetected virus production.
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http://dx.doi.org/10.4049/jimmunol.0803218DOI Listing
February 2009

Synthesis and biological evaluation of a chitobiose-based peptide N-glycanase inhibitor library.

J Org Chem 2009 Jan;74(2):605-16

Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.

Peptide N-glycanase (PNGase), the enzyme responsible for the deglycosylation of N-linked glycoproteins, has an active site related to that of cysteine proteases. Chitiobiose was equipped with electrophilic traps often used in cysteine protease inhibitors, and the resulting compounds were evaluated as PNGase inhibitors. We found that the electrophilic trap of the inhibitor has a great influence on the potency of the compounds with the chloromethyl ketone inhibitor being the first potent C-glycoside-based PNGase inhibitor.
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http://dx.doi.org/10.1021/jo801906sDOI Listing
January 2009

Epstein-Barr virus evasion of CD8(+) and CD4(+) T cell immunity via concerted actions of multiple gene products.

Semin Cancer Biol 2008 Dec 25;18(6):397-408. Epub 2008 Oct 25.

Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center (LUMC), Leiden, The Netherlands.

Upon primary infection, EBV establishes a latent infection in B cells, characterized by maintenance of the viral genome in the absence of viral replication. The Epstein-Barr Nuclear Antigen 1 (EBNA1) plays a crucial role in maintenance of the viral DNA episome and is consistently expressed in all EBV-associated malignancies. Compared to other EBV latent gene products, EBNA1 is poorly recognized by CD8(+) T lymphocytes. Recent studies are discussed that shed new light on the mechanisms that underlie this unusual lack of CD8(+) T cell activation. Whereas the latent phase is characterized by the expression of a limited subset of viral gene products, the full repertoire of over 80 EBV lytic gene products is expressed during the replicative phase. Despite this abundance of potential T cell antigens, which indeed give rise to a strong response of CD4(+) and CD8(+) T lymphocytes, the virus can replicate successfully. Evidence is accumulating that this paradoxical situation is the result of actions of multiple viral gene products, inhibiting discrete stages of the MHC class I and class II antigen presentation pathways. Immediately after initiation of the lytic cycle, BNLF2a prevents peptide-loading of MHC class I molecules through inhibition of the Transporter associated with Antigen Processing, TAP. This will reduce presentation of viral antigens by the large ER-resident pool of MHC class I molecules. Synthesis of new MHC class I molecules is blocked by BGLF5. Viral-IL10 causes a reduction in mRNA levels of TAP1 and bli/LMP2, a subunit of the immunoproteasome. MHC class I molecules present at the cell surface are downregulated by BILF1. Also the antigen presenting capacity of MHC class II molecules is severely compromised by multiple EBV lytic gene products, including gp42/gH/gL, BGLF5, and vIL-10. In this review, we discuss how concerted actions of these EBV lytic proteins result in highly effective interference with CD8(+) and CD4(+) T cell surveillance, thereby providing the virus with a window for undisturbed generation of viral progeny.
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http://dx.doi.org/10.1016/j.semcancer.2008.10.008DOI Listing
December 2008

A CD8+ T cell immune evasion protein specific to Epstein-Barr virus and its close relatives in Old World primates.

J Exp Med 2007 Aug 9;204(8):1863-73. Epub 2007 Jul 9.

Cancer Research UK Institute for Cancer Studies and MRC Centre for Immune Regulation, University of Birmingham, Edgbaston, Birmingham, B15 2TT, England, UK.

gamma 1-Herpesviruses such as Epstein-Barr virus (EBV) have a unique ability to amplify virus loads in vivo through latent growth-transforming infection. Whether they, like alpha- and beta-herpesviruses, have been driven to actively evade immune detection of replicative (lytic) infection remains a moot point. We were prompted to readdress this question by recent work (Pudney, V.A., A.M. Leese, A.B. Rickinson, and A.D. Hislop. 2005. J. Exp. Med. 201:349-360; Ressing, M.E., S.E. Keating, D. van Leeuwen, D. Koppers-Lalic, I.Y. Pappworth, E.J.H.J. Wiertz, and M. Rowe. 2005. J. Immunol. 174:6829-6838) showing that, as EBV-infected cells move through the lytic cycle, their susceptibility to EBV-specific CD8(+) T cell recognition falls dramatically, concomitant with a reductions in transporter associated with antigen processing (TAP) function and surface human histocompatibility leukocyte antigen (HLA) class I expression. Screening of genes that are unique to EBV and closely related gamma 1-herpesviruses of Old World primates identified an early EBV lytic cycle gene, BNLF2a, which efficiently blocks antigen-specific CD8(+) T cell recognition through HLA-A-, HLA-B-, and HLA-C-restricting alleles when expressed in target cells in vitro. The small (60-amino acid) BNLF2a protein mediated its effects through interacting with the TAP complex and inhibiting both its peptide- and ATP-binding functions. Furthermore, this targeting of the major histocompatibility complex class I pathway appears to be conserved among the BNLF2a homologues of Old World primate gamma 1-herpesviruses. Thus, even the acquisition of latent cycle genes endowing unique growth-transforming ability has not liberated these agents from evolutionary pressure to evade CD8(+) T cell control over virus replicative foci.
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http://dx.doi.org/10.1084/jem.20070256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2118677PMC
August 2007

Cdc37p is required for stress-induced high-osmolarity glycerol and protein kinase C mitogen-activated protein kinase pathway functionality by interaction with Hog1p and Slt2p (Mpk1p).

Eukaryot Cell 2007 Mar 12;6(3):521-32. Epub 2007 Jan 12.

Department of Biochemistry and Molecular Biology, Faculty of Science, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.

The yeast Saccharomyces cerevisiae utilizes rapidly responding mitogen-activated protein kinase (MAPK) signaling cascades to adapt efficiently to a changing environment. Here we report that phosphorylation of Cdc37p, an Hsp90 cochaperone, by casein kinase 2 controls the functionality of two MAPK cascades in yeast. These pathways, the high-osmolarity glycerol (HOG) pathway and the cell integrity (protein kinase C) MAPK pathway, mediate adaptive responses to high osmotic and cell wall stresses, respectively. Mutation of the phosphorylation site Ser14 in Cdc37p renders cells sensitive to osmotic stress and cell wall perturbation by calcofluor white. We found that levels of the MAPKs Hog1p and Slt2p (Mpk1p) in cells are reduced in a cdc37-S14A mutant, and consequently downstream responses mediated by Hog1p and Slt2p are compromised. Furthermore, we present evidence that Hog1p and Slt2p both interact in a complex with Cdc37p in vivo, something that has not been reported previously. The interaction of Hsp90, Slt2p, and Hog1p with Cdc37p depends on the phosphorylation status of Cdc37p. In fact, our biochemical data show that the osmosensitive phenotype of the cdc37-S14A mutant is due to the loss of the interaction between Cdc37p, Hog1p, and Hsp90. Likewise, during cell wall stress, the interaction of Slt2p with Cdc37p and Hsp90 is crucial for Slt2p-dependent downstream responses, such as the activation of the transcription factor Rlm1p. Interestingly, phosphorylated Slt2p, but not phosphorylated Hog1p, has an increased affinity for Cdc37p. Together these observations suggest that Cdc37p acts as a regulator of MAPK signaling.
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http://dx.doi.org/10.1128/EC.00343-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1828922PMC
March 2007