RESUMO
Recurrent, ancient arms races between viruses and hosts have shaped both host immunological defense strategies as well as viral countermeasures. One such battle is waged by the glycoprotein US11 encoded by the persisting human cytomegalovirus. US11 mediates degradation of major histocompatibility class I (MHC-I) molecules to prevent CD8+ T-cell activation. Here, we studied the consequences of the arms race between US11 and primate MHC-A proteins, leading us to uncover a tit-for-tat coevolution and its impact on MHC-A diversification. We found that US11 spurred MHC-A adaptation to evade viral antagonism: In an ancestor of great apes, the MHC-A A2 lineage acquired a Pro184Ala mutation, which confers resistance against the ancestral US11 targeting strategy. In response, US11 deployed a unique low-complexity region (LCR), which exploits the MHC-I peptide loading complex to target the MHC-A2 peptide-binding groove. In addition, the global spread of the human HLA-A*02 allelic family prompted US11 to employ a superior LCR strategy with an optimally fitting peptide mimetic that specifically antagonizes HLA-A*02. Thus, despite cytomegaloviruses low pathogenic potential, the increasing commitment of US11 to MHC-A has significantly promoted diversification of MHC-A in hominids.
Assuntos
Antígenos de Histocompatibilidade Classe I , Hominidae , Animais , Humanos , Proteínas Virais/metabolismo , Citomegalovirus , Hominidae/genética , Hominidae/metabolismo , Linhagem Celular , Antígenos de Histocompatibilidade/metabolismo , Antígenos HLA-A/metabolismo , Peptídeos/metabolismoRESUMO
ABSTRACT: Human cytomegalovirus (HCMV) reactivation poses a substantial risk to patients receiving tranplants. Effective risk stratification and vaccine development is hampered by a lack of HCMV-derived immunogenic peptides in patients with common HLA-A∗03:01 and HLA-B∗15:01 haplotypes. This study aimed to discover novel HCMV immunogenic peptides for these haplotypes by combining ribosome sequencing (Ribo-seq) and mass spectrometry with state-of-the-art computational tools, Peptide-PRISM and Probabilistic Inference of Codon Activities by an EM Algorithm. Furthermore, using machine learning, an algorithm was developed to predict immunogenicity based on translational activity, binding affinity, and peptide localization within small open reading frames to identify the most promising peptides for in vitro validation. Immunogenicity of these peptides was subsequently tested by analyzing peptide-specific T-cell responses of HCMV-seropositive and -seronegative healthy donors as well as patients with transplants. This resulted in the direct identification of 3 canonical and 1 cryptic HLA-A∗03-restricted immunogenic peptides as well as 5 canonical and 1 cryptic HLA-B∗15-restricted immunogenic peptide, with a specific interferon gamma-positive (IFN-γ+)/CD8+ T-cell response of ≥0.02%. High T-cell responses were detected against 2 HLA-A∗03-restricted and 3 HLA-B∗15-restricted canonical peptides with frequencies of up to 8.77% IFN-γ+/CD8+ T cells in patients after allogeneic stem cell transplantation. Therefore, our comprehensive strategy establishes a framework for efficient identification of novel immunogenic peptides from both existing and novel Ribo-seq data sets.
Assuntos
Citomegalovirus , Epitopos de Linfócito T , Humanos , Peptídeos , Antígenos HLA-B , Antígenos HLA-ARESUMO
A single model system for integrative studies on multiple facets of antigen presentation is lacking. PAKC is a novel panel of ten cell lines knocked out for individual components of the HLA class I antigen presentation pathway. PAKC will accelerate HLA-I research in the fields of oncology, infectiology, and autoimmunity.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Autoimunidade/imunologia , Humanos , Neoplasias/imunologia , Transdução de Sinais/imunologiaRESUMO
HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Linfócitos T CD8-Positivos/imunologia , Glioma/imunologia , Glicoesfingolipídeos/metabolismo , Glicosiltransferases/metabolismo , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoterapia/métodos , Apresentação de Antígeno , Ácido Aspártico Endopeptidases/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioma/mortalidade , Glicoesfingolipídeos/imunologia , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Ativação Linfocitária , Transdução de Sinais , Análise de Sobrevida , Evasão TumoralRESUMO
CMVs efficiently target MHC I molecules to avoid recognition by cytotoxic T cells. However, the lack of MHC I on the cell surface renders the infected cell susceptible to NK cell killing upon missing self recognition. To counter this, mouse CMV (MCMV) rescues some MHC I molecules to engage inhibitory Ly49 receptors. Here we identify a new viral protein, MATp1, that is essential for MHC I surface rescue. Rescued altered-self MHC I molecules show increased affinity to inhibitory Ly49 receptors, resulting in inhibition of NK cells despite substantially reduced MHC I surface levels. This enables the virus to evade recognition by licensed NK cells. During evolution, this novel viral immune evasion mechanism could have prompted the development of activating NK cell receptors that are specific for MATp1-modified altered-self MHC I molecules. Our study solves a long-standing conundrum of how MCMV avoids recognition by NK cells, unravels a fundamental new viral immune evasion mechanism, and demonstrates how this forced the evolution of virus-specific activating MHC I-restricted Ly49 receptors.
Assuntos
Infecções por Herpesviridae/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Evasão da Resposta Imune/imunologia , Células Matadoras Naturais/imunologia , Muromegalovirus/metabolismo , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Proteínas Virais/metabolismo , Animais , Antígenos Ly/genética , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Fibroblastos/metabolismo , Infecções por Herpesviridae/virologia , Antígenos de Histocompatibilidade Classe I/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor 1 Desencadeador da Citotoxicidade Natural/genéticaRESUMO
Herpesvirus infections are highly prevalent in the human population and persist for life. They are often acquired subclinically but potentially progress to life-threatening diseases in immunocompromised individuals. The interferon system is indispensable for the control of herpesviral replication. However, the responsible antiviral effector mechanisms are not well characterized. The type I interferon-induced, human myxovirus resistance 2 (MX2) gene product MxB, a dynamin-like large GTPase, has recently been identified as a potent inhibitor of HIV-1. We now show that MxB also interferes with an early step of herpesvirus replication, affecting alpha-, beta-, and gammaherpesviruses before or at the time of immediate early gene expression. Defined MxB mutants influencing GTP binding and hydrolysis revealed that the effector mechanism against herpesviruses is thoroughly different from that against HIV-1. Overall, our findings demonstrate that MxB serves as a broadly acting intracellular restriction factor that controls the establishment of not only retrovirus but also herpesvirus infection of all three subfamilies.IMPORTANCE Human herpesviruses pose a constant threat to human health. Reactivation of persisting herpesvirus infections, particularly in immunocompromised individuals and the elderly, can cause severe diseases, such as zoster, pneumonia, encephalitis, or cancer. The interferon system is relevant for the control of herpesvirus replication as exemplified by fatal disease outcomes in patients with primary immunodeficiencies. Here, we describe the interferon-induced, human MX2 gene product MxB as an efficient restriction factor of alpha-, beta-, and gammaherpesviruses. MxB has previously been described as an inhibitor of HIV-1. Importantly, our mutational analyses of MxB reveal an antiviral mechanism of herpesvirus restriction distinct from that against HIV-1. Thus, the dynamin-like MxB GTPase serves as a broadly acting intracellular restriction factor that controls retrovirus as well as herpesvirus infections.
Assuntos
Infecções por Herpesviridae/prevenção & controle , Herpesviridae/fisiologia , Mutação , Proteínas de Resistência a Myxovirus/genética , Replicação Viral/genética , Células A549 , Herpesviridae/genética , Infecções por Herpesviridae/virologia , Humanos , Imunidade Inata , Interferons , Proteínas de Resistência a Myxovirus/imunologia , Replicação Viral/imunologiaRESUMO
Ribosome profiling has been used to predict thousands of short open reading frames (sORFs) in eukaryotic cells, but it suffers from substantial levels of noise. PRICE (https://github.com/erhard-lab/price) is a computational method that models experimental noise to enable researchers to accurately resolve overlapping sORFs and noncanonical translation initiation. We experimentally validated translation using major histocompatibility complex class I (MHC I) peptidomics and observed that sORF-derived peptides efficiently enter the MHC I presentation pathway and thus constitute a substantial fraction of the antigen repertoire.
Assuntos
Biologia Computacional , Peptídeos/metabolismo , Proteômica/métodos , Ribossomos/fisiologia , Genes MHC Classe I , Modelos Biológicos , Biossíntese de Proteínas , Pegadas de Proteínas , SoftwareRESUMO
The transporter associated with antigen processing (TAP) translocates antigenic peptides into the endoplasmic reticulum (ER) lumen for loading onto MHC class I molecules. This is a key step in the control of viral infections through CD8+ T-cells. The herpes simplex virus type-1 encodes an 88 amino acid long species-specific TAP inhibitor, ICP47, that functions as a high affinity competitor for the peptide binding site on TAP. It has previously been suggested that the inhibitory function of ICP47 resides within the N-terminal region (residues 1-35). Here we show that mutation of the highly conserved 50PLL52 motif within the central region of ICP47 attenuates its inhibitory capacity. Taking advantage of the human cytomegalovirus-encoded TAP inhibitor US6 as a luminal sensor for conformational changes of TAP, we demonstrated that the 50PLL52 motif is essential for freezing of the TAP conformation. Moreover, hierarchical functional interaction sites on TAP dependent on 50PLL52 could be defined using a comprehensive set of human-rat TAP chimeras. This data broadens our understanding of the molecular mechanism underpinning TAP inhibition by ICP47, to include the 50PLL52 sequence as a stabilizer that tethers the TAP-ICP47 complex in an inward-facing conformation.
Assuntos
Sequência de Aminoácidos , Sequência Conservada , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/metabolismo , Proteínas Virais/antagonistas & inibidores , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Transporte Proteico , Ratos , Relação Estrutura-Atividade , Proteínas Virais/químicaRESUMO
Infection with adenovirus is a major cause of infectious mortality in children following hematopoietic stem-cell transplantation. While adoptive transfer of epitope-specific T cells is a particularly effective therapeutic approach, there are few suitable adenoviral peptide epitopes described to date. Here, we describe the adenoviral peptide epitope FRKDVNMVL from hexon protein, and its variant FRKDVNMIL, that is restricted by human leukocyte antigen (HLA)-C*0702. Since HLA-C*0702 can be recognized by both T cells and natural killer (NK) cells, we characterized responses by both cell types. T cells specific for FRKDVNMVL were detected in peripheral blood mononuclear cells expanded from eight of ten healthy HLA-typed donors by peptide-HLA multimer staining, and could also be detected by cultured interferon γ ELISpot assays. Surprisingly, HLA-C*0702 was not downregulated during infection, in contrast to the marked downregulation of HLA-A*0201, suggesting that adenovirus cannot evade T cell responses to HLA-C*0702-restricted peptide epitopes. By contrast, NK responses were inhibited following adenoviral peptide presentation. Notably, presentation of the FRKDVNMVL peptide enhanced binding of HLA-C*0702 to the inhibitory receptor KIR2DL3 and decreased NK cytotoxic responses, suggesting that adenoviruses may use this peptide to evade NK responses. Given the immunodominance of FRKDVNMVL-specific T cell responses, apparent lack of HLA-C*0702 downregulation during infection, and the high frequency of this allotype, this peptide epitope may be particularly useful for adoptive T cell transfer therapy of adenovirus infection.
Assuntos
Adenovírus Humanos/imunologia , Apresentação de Antígeno , Epitopos de Linfócito T/metabolismo , Antígenos HLA-C/metabolismo , Evasão da Resposta Imune , Células Matadoras Naturais/imunologia , Linfócitos T/imunologia , Epitopos de Linfócito T/imunologia , HumanosRESUMO
The receptor-like protein tyrosine phosphatase CD45 is expressed on the surface of cells of hematopoietic origin and has a pivotal role for the function of these cells in the immune response. Here we report that following infection of macrophages with mouse cytomegalovirus (MCMV) the cell surface expression of CD45 is drastically diminished. Screening of a set of MCMV deletion mutants allowed us to identify the viral gene m42 of being responsible for CD45 down-modulation. Moreover, expression of m42 independent of viral infection upon retroviral transduction of the RAW264.7 macrophage cell line led to comparable regulation of CD45 expression. In immunocompetent mice infected with an m42 deletion mutant lower viral titers were observed in all tissues examined when compared to wildtype MCMV, indicating an important role of m42 for viral replication in vivo. The m42 gene product was identified as an 18 kDa protein expressed with early kinetics and is predicted to be a tail-anchored membrane protein. Tracking of surface-resident CD45 molecules revealed that m42 induces internalization and degradation of CD45. The observation that the amounts of the E3 ubiquitin ligases Itch and Nedd4 were diminished in cells expressing m42 and that disruption of a PY motif in the N-terminal part of m42 resulted in loss of function, suggest that m42 acts as an activator or adaptor for these Nedd4-like ubiquitin ligases, which mark CD45 for lysosomal degradation. In conclusion, the down-modulation of CD45 expression in MCMV-infected myeloid cells represents a novel pathway of virus-host interaction.
Assuntos
Regulação Viral da Expressão Gênica/genética , Genes Virais/genética , Infecções por Herpesviridae/genética , Antígenos Comuns de Leucócito/biossíntese , Macrófagos/virologia , Animais , Regulação para Baixo , Citometria de Fluxo , Imunofluorescência , Células HEK293 , Infecções por Herpesviridae/metabolismo , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Muromegalovirus , Células RAW 264.7RESUMO
The existence and expansion of adaptive NK-cell subsets have been linked to HCMV infection. Phenotypically, a majority of adaptive NK cells expresses the activating receptor NKG2C and CD57. Some of the molecular factors driving the expansion of NKG2C+ CD57+ NK cells in HCMV infection have been identified. The direct interaction of adaptive NK cells with HCMV-infected cells, preceding the expansion, however, remains less studied. Recently, adaptive NK cells were reported to express higher levels of the co-activating receptor CD2. We explored whether CD2 was directly involved in the response of adaptive NK cells to HCMV. In a co-culture system of human PBMCs and productively infected fibroblasts, we observed an upregulation of CD69, CD25, and HLA-DR on all NK cells. However, only in adaptive NK cells was this increase largely blocked by antibodies against CD2 and CD58. Functionally, this blockade also resulted in diminished production of IFN-γ and TNF-α by adaptive human NK cells in response to HCMV-infected cells. Our results demonstrate that binding of CD2 to upregulated CD58 on infected cells is a critical event for antibody-mediated activation and subsequent effector functions of adaptive NKG2C+ CD57+ NK cells during the antiviral response.
Assuntos
Antígenos CD2/metabolismo , Antígenos CD58/metabolismo , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Fibroblastos/imunologia , Células Matadoras Naturais/imunologia , Imunidade Adaptativa , Anticorpos/metabolismo , Proliferação de Células , Células Cultivadas , Fibroblastos/virologia , Humanos , Interferon gama/metabolismo , Ativação Linfocitária , Ligação Proteica , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Cells in our body can induce hundreds of antiviral genes following virus sensing, many of which remain largely uncharacterized. CEACAM1 has been previously shown to be induced by various innate systems; however, the reason for such tight integration to innate sensing systems was not apparent. Here, we show that CEACAM1 is induced following detection of HCMV and influenza viruses by their respective DNA and RNA innate sensors, IFI16 and RIG-I. This induction is mediated by IRF3, which bound to an ISRE element present in the human, but not mouse, CEACAM1 promoter. Furthermore, we demonstrate that, upon induction, CEACAM1 suppresses both HCMV and influenza viruses in an SHP2-dependent process and achieves this broad antiviral efficacy by suppressing mTOR-mediated protein biosynthesis. Finally, we show that CEACAM1 also inhibits viral spread in ex vivo human decidua organ culture.
Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Citomegalovirus/fisiologia , Orthomyxoviridae/fisiologia , Animais , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Proteína DEAD-box 58/metabolismo , DNA Viral/metabolismo , Humanos , Influenza Humana/metabolismo , Influenza Humana/virologia , Fator Regulador 3 de Interferon/metabolismo , Camundongos , Técnicas de Cultura de Órgãos , Biossíntese de Proteínas , Receptores Imunológicos , Serina-Treonina Quinases TOR/metabolismo , Replicação ViralRESUMO
In the presence of the murine cytomegalovirus (mCMV) gp40 (m152) protein, murine major histocompatibility complex (MHC) class I molecules do not reach the cell surface but are retained in an early compartment of the secretory pathway. We find that gp40 does not impair the folding or high-affinity peptide binding of the class I molecules but binds to them, leading to their retention in the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, most likely by retrieval from the cis-Golgi to the ER. We identify a sequence in gp40 that is required for both its own retention in the early secretory pathway and for that of class I molecules.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Muromegalovirus/metabolismo , Via Secretória , Proteínas Virais/metabolismo , Animais , Camundongos , Modelos Biológicos , Peptídeos/metabolismo , Ligação ProteicaRESUMO
Human cytomegalovirus (HCMV) infection is the most common cause of congenital viral infections and a major source of morbidity and mortality after organ transplantation. NK cells are pivotal effector cells in the innate defense against CMV. Recently, hallmarks of adaptive responses, such as memory-like features, have been recognized in NK cells. HCMV infection elicits the expansion of an NK cell subset carrying an activating receptor heterodimer, comprising CD94 and NKG2C (CD94/NKG2C), a response that resembles the clonal expansion of adaptive immune cells. Here, we determined that expansion of this NKG2C(+) subset and general NK cell recovery rely on signals derived from CD14(+) monocytes. In a coculture system, a subset of CD14(+) cells with inflammatory monocyte features produced IL-12 in response to HCMV-infected fibroblasts, and neutralization of IL-12 in this model substantially reduced CD25 upregulation and NKG2C(+) subset expansion. Finally, blockade of CD94/NKG2C on NK cells or silencing of the cognate ligand HLA-E in infected fibroblasts greatly impaired expansion of NKG2C(+) NK cells. Together, our results reveal that IL-12, CD14(+) cells, and the CD94/NKG2C/HLA-E axis are critical for the expansion of NKG2C(+) NK cells in response to HCMV infection. Moreover, strategies targeting the NKG2C(+) NK cell subset have the potential to be exploited in NK cell-based intervention strategies against viral infections and cancer.
Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Interleucina-12/imunologia , Células Matadoras Naturais/imunologia , Monócitos/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Infecções por Citomegalovirus/patologia , Feminino , Humanos , Imunidade Celular , Subunidade alfa de Receptor de Interleucina-2/imunologia , Células Matadoras Naturais/patologia , Receptores de Lipopolissacarídeos/imunologia , Masculino , Monócitos/patologia , Subfamília D de Receptores Semelhantes a Lectina de Células NK/imunologia , Antígenos HLA-ERESUMO
Herpesviruses escape elimination by cytotoxic T lymphocytes through specific interference with the antigen-presenting function of MHC class I (MHC I) molecules. The transporter associated with antigen processing (TAP) forms a bottleneck in the MHC I antigen presentation pathway. The fact that multiple viruses, especially herpesviruses, encode molecules blocking TAP function is a case in point. The action of these viral immuno evasins is usually potent and very specific, making these proteins valuable tools for studying the cell biology of antigen presentation, including alternative antigen processing pathways. Yet, no dedicated TAP inhibitor has been described for any of the mouse herpesviruses. To permit the use of immuno evasins derived from non-mouse herpesviruses in mouse models, we assessed the cross-species activity of four TAP inhibitors and one tapasin inhibitor in the context of three different mouse haplotypes, H-2(b), H-2(d), and H-2(k). Two of the four TAP inhibitors, the bovine herpesvirus 1-encoded UL49.5 and the human cytomegalovirus (HCMV)-encoded US6 protein, potently inhibited mouse TAP. ICP47 and BNLF2a, encoded by herpes simplexvirus 1 and Epstein-Barr virus, respectively, failed to inhibit TAP in all mouse cells tested. Previous work, however, demonstrated that US6 did not cross the mouse species barrier. We now show that substitution of the cysteine residue at position 108 was responsible for this lack of activity. The HCMV-encoded tapasin inhibitor US3 efficiently downregulated H-2(d) molecules on 3T3 cells, but not in other cell lines tested. Finally, we show that synthetic peptides comprising the functional domain of US6 can be exploited as a versatile TAP inhibitor. In conclusion, a complete overview is presented of the applicability of herpesvirus-encoded TAP and tapasin inhibitors in mouse cells of different genetic background.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Herpesviridae/imunologia , Evasão da Resposta Imune/imunologia , Proteínas Virais/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/imunologia , Sequência de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Bovinos , Cisteína/metabolismo , Regulação para Baixo/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Haplótipos/genética , Células HeLa , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Evasão da Resposta Imune/efeitos dos fármacos , Interferon gama/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Peptídeos/química , Peptídeos/farmacologia , Estrutura Terciária de Proteína , Especificidade da EspécieRESUMO
The endoplasmic reticulum-resident human cytomegalovirus glycoprotein US6 (gpUS6) inhibits peptide translocation by the transporter associated with antigen processing (TAP) to prevent loading of major histocompatibility complex class I molecules and antigen presentation to CD8+ T cells. TAP is formed by two subunits, TAP1 and TAP2, each containing one multispanning transmembrane domain (TMD) and a cytosolic nucleotide binding domain. Here we reported that the blockade of TAP by gpUS6 is species-restricted, i.e. gpUS6 inhibits human TAP but not rat TAP. Co-expression of human and rat subunits of TAP demonstrates independent binding of gpUS6 to human TAP1 and TAP2, whereas gpUS6 does not bind to rat TAP subunits. gpUS6 associates with preformed TAP1/2 heterodimers but not with unassembled TAP subunits. To locate domains of TAP required for gpUS6 binding and function, we took advantage of reciprocal human/rat intrachain TAP chimeras. Each TAP subunit forms two contact sites within its TMD interacting with gpUS6. The dominant gpUS6-binding site on TAP2 maps to an N-terminal loop, whereas inhibition of peptide transport is mediated by a C-terminal loop of the TMD. For TAP1, two gpUS6 binding domains are formed by loops of the C-terminal TMD. The domain required for TAP inactivation is built by a distal loop of the C-terminal TMD, indicating a topology of TAP1 comprising 10 endoplasmic reticulum transmembrane segments. By forming multimeric complexes, gpUS6 reaches the distant target domains to arrest peptide transport. The data revealed a nonanalogous multipolar bridging of the TAP TMDs by gpUS6.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Subunidades Proteicas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Transportadores de Cassetes de Ligação de ATP , Sequência de Aminoácidos , Animais , Transporte Biológico , Linhagem Celular , Dimerização , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Dados de Sequência Molecular , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Proteínas de Ligação a RNA/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Proteínas Virais/genéticaRESUMO
Measles virus (MV) infection and vaccination induce long-lasting immunity and neutralizing-antibody responses that are directed against the MV haemagglutinin (H) and the fusion (F) protein. A new MV genotype, D7, emerged recently in western Germany and rapidly replaced the long-term endemically circulating genotypes C2 and D6. Analysis of the H gene of C2, D6, D7 and vaccine viruses revealed uniform sequences for each genotype. Interestingly, a consistent exchange of seven distinct amino acids in the D7 H was observed when compared with residues shared between C2, D6 and vaccine viruses, and one exchange (D416-->N) in the D7 H was associated with an additional N-linked glycosylation. In contrast, the F gene is highly conserved between MVs of these genotypes. To test whether the D7 H protein escapes from antibody responses that were raised against earlier circulating or vaccine viruses, the neutralizing capacity of mAbs recognizing seven distinct domains on the H of an Edmonston-related MV was compared. The mAbs revealed a selective and complete loss of two neutralizing epitopes on the D7 H when compared with C2, D6 and vaccine viruses. To assess whether these alterations of the D7 H affect the neutralizing capacity of polyclonal B-cell responses, genotype-specific antisera were produced in cotton rats. However, no significant genotype-dependent difference was found. Likewise, human sera obtained from vaccinees (n=7) and convalescents (n=6) did not distinguish between the MV genotypes. Although the hypothesis of selection of D7 viruses by pre-existing neutralizing antibodies is compatible with the differing pattern of neutralizing epitopes on the H protein, it was not confirmed by the results of MV neutralization with polyclonal sera.
Assuntos
Vírus do Sarampo/imunologia , Sarampo/imunologia , Proteínas Virais/imunologia , Adolescente , Adulto , Animais , Anticorpos Antivirais/imunologia , Criança , Pré-Escolar , Convalescença , Genótipo , Alemanha , Humanos , Soros Imunes/imunologia , Lactente , Vacina contra Sarampo/imunologia , Vírus do Sarampo/genética , Pessoa de Meia-Idade , Testes de Neutralização , Ratos , Vacinação , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologiaRESUMO
The high incidence of hepatitis C virus (HCV) persistence raises the question of how HCV interferes with host immune responses. Studying a single-source HCV outbreak, we identified an HCV mutation that impaired correct carboxyterminal cleavage of an immunodominant HLA-A2-restricted CD8 cell epitope that is frequently recognized by recovered patients. The mutation, a conservative HCV nonstructural protein 3 (NS3) tyrosine to phenylalanine substitution, was absent in 54 clones of the infectious source, but present in 15/21 (71%) HLA-A2-positive and in 11/24 (46%) HLA-A2-negative patients with chronic hepatitis C. In order to analyze whether the mutation affected the processing of the HLA-A2-restricted CD8 cell epitope, mutant and wild-type NS3 polypeptides were digested in vitro with 20S constitutive proteasomes and with immunoproteasomes. The presence of the mutation resulted in impaired carboxyterminal cleavage of the epitope. In order to analyze whether impaired epitope processing affected T cell priming in vivo, HLA-A2-transgenic mice were infected with vaccinia viruses encoding either wild-type or mutant HCV NS3. The mutant induced fewer epitope-specific, IFN-gamma;-producing and fewer tetramer(+) cells than the wild type. These data demonstrate how a conservative mutation in the flanking region of an HCV epitope impairs the induction of epitope-specific CD8(+) T cells and reveal a mechanism that may contribute to viral sequence evolution in infected patients.