RESUMO
Coevolution of herpesviruses and their hosts has driven the development of both host antiviral mechanisms to detect and eliminate infected cells and viral ploys to escape immune surveillance. Among the immune-evasion strategies used by the lymphocryptovirus (γ(1)-herpesvirus) EBV is the downregulation of surface HLA class I expression by the virally encoded G protein-coupled receptor BILF1, thereby impeding presentation of viral Ags and cytotoxic T cell recognition of the infected cell. In this study, we show EBV BILF1 to be expressed early in the viral lytic cycle. BILF1 targets a broad range of HLA class I molecules, including multiple HLA-A and -B types and HLA-E. In contrast, HLA-C was only marginally affected. We advance the mechanistic understanding of the process by showing that the cytoplasmic C-terminal tail of EBV BILF1 is required for reducing surface HLA class I expression. Susceptibility to BILF1-mediated downregulation, in turn, is conferred by specific residues in the intracellular tail of the HLA class I H chain. Finally, we explore the evolution of BILF1 within the lymphocryptovirus genus. Although the homolog of BILF1 encoded by the lymphocryptovirus infecting Old World rhesus primates shares the ability of EBV to downregulate cell surface HLA class I expression, this function is not possessed by New World marmoset lymphocryptovirus BILF1. Therefore, this study furthers our knowledge of the evolution of immunoevasive functions by the lymphocryptovirus genus of herpesviruses.
Assuntos
Citoplasma/imunologia , Regulação para Baixo/imunologia , Evolução Molecular , Herpesvirus Humano 4/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Receptores Acoplados a Proteínas G/fisiologia , Proteínas Virais/fisiologia , Alelos , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Citoplasma/metabolismo , Citoplasma/virologia , Regulação Viral da Expressão Gênica/imunologia , Marcação de Genes , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Evasão da Resposta Imune , Glicoproteínas de Membrana/biossíntese , Fragmentos de Peptídeos/fisiologia , Transdução de Sinais/imunologiaRESUMO
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.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Regulação da Expressão Gênica , Herpesvirus Humano 4/metabolismo , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/metabolismo , Cercopithecidae , Clonagem Molecular , Citometria de Fluxo , Antígenos HLA/química , Antígenos HLA/metabolismo , Herpesviridae/metabolismo , Humanos , Sistema Imunitário/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Desoxirribonucleases/química , Desoxirribonucleases/metabolismo , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/enzimologia , Herpesvirus Humano 4/fisiologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular , Desoxirribonucleases/genética , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/química , Herpesvirus Humano 4/genética , Humanos , Dados de Sequência Molecular , Biossíntese de Proteínas , Estabilidade de RNA , Alinhamento de Sequência , Proteínas Virais/genética , Replicação ViralRESUMO
Viruses use a wide range of strategies to modulate the host immune response. The human gammaherpesvirus EBV, causative agent of infectious mononucleosis and several malignant tumors, encodes proteins that subvert immune responses, notably those mediated by T cells. Less is known about EBV interference with innate immunity, more specifically at the level of TLR-mediated pathogen recognition. The viral dsDNA sensor TLR9 is expressed on B cells, a natural target of EBV infection. Here, we show that EBV particles trigger innate immune signaling pathways through TLR9. Furthermore, using an in vitro system for productive EBV infection, it has now been possible to compare the expression of TLRs by EBV(-) and EBV(+) human B cells during the latent and lytic phases of infection. Several TLRs were found to be differentially expressed either in latently EBV-infected cells or after induction of the lytic cycle. In particular, TLR9 expression was profoundly decreased at both the RNA and protein levels during productive EBV infection. We identified the EBV lytic-phase protein BGLF5 as a protein that contributes to downregulating TLR9 levels through RNA degradation. Reducing the levels of a pattern-recognition receptor capable of sensing the presence of EBV provides a mechanism by which the virus could obstruct host innate antiviral responses.
Assuntos
Desoxirribonucleases/fisiologia , Regulação para Baixo/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/imunologia , Receptor Toll-Like 9/antagonistas & inibidores , Receptor Toll-Like 9/biossíntese , Proteínas Virais/fisiologia , Latência Viral/imunologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/patologia , Subpopulações de Linfócitos B/virologia , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/patologia , Linfoma de Burkitt/virologia , Linhagem Celular Tumoral , Células Cultivadas , Regulação para Baixo/genética , Infecções por Vírus Epstein-Barr/metabolismo , Regulação Viral da Expressão Gênica/imunologia , Células HEK293 , Herpesvirus Humano 4/patogenicidade , Humanos , RNA Viral/antagonistas & inibidores , RNA Viral/metabolismo , Receptor Toll-Like 9/genética , Vírion/imunologia , Ativação Viral/imunologiaRESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/imunologia , Apresentação de Antígeno/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/fisiologia , Antígenos de Histocompatibilidade Classe I/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Infecções por Vírus Epstein-Barr/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução Genética , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
The DNase/alkaline exonuclease (AE) genes are well conserved in all herpesvirus families, but recent studies have shown that the AE proteins of gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) exhibit an additional function which shuts down host protein synthesis. One correlate of this additional shutoff function is that levels of cell surface HLA molecules are downregulated, raising the possibility that shutoff/AE genes of gammaherpesviruses might contribute to viral immune evasion. In this study, we show that both BGLF5 (EBV) and SOX (KSHV) shutoff/AE proteins do indeed impair the ability of virus-specific CD8+ T-cell clones to recognize endogenous antigen via HLA class I. Random mutagenesis of the BGLF5 gene enabled us to genetically separate the shutoff and AE functions and to demonstrate that the shutoff function was the critical factor determining whether BGLF5 mutants can impair T-cell recognition. These data provide further evidence that EBV has multiple mechanisms to modulate HLA class I-restricted T-cell responses, thus enabling the virus to replicate and persist in the immune-competent host.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Desoxirribonucleases/metabolismo , Gammaherpesvirinae/enzimologia , Linhagem Celular , Desoxirribonucleases/genética , Imunofluorescência , Gammaherpesvirinae/imunologia , Gammaherpesvirinae/fisiologia , Humanos , Proteínas Virais/genética , Replicação ViralRESUMO
Herpesviruses are known to influence expression of major histocompatibility complex (MHC) class I molecules on the surface of infected cells using a variety of mechanisms. Downregulation of MHC class I expression prohibits detection and elimination of infected cells by cytotoxic T lymphocytes. To investigate the effect of rat cytomegalovirus (RCMV) infection on MHC class I expression, we infected immortalized and primary rat fibroblasts with RCMV and monitored surface expression of MHC class I molecules at various time-points postinfection. These experiments revealed a downregulation of MHC class I surface expression by RCMV, a phenomenon that has also been reported for human and murine CMV. However, in contrast to the other cytomegaloviruses, RCMV causes only a temporal downregulation of MHC class I, with a maximal decrease at 12 h postinfection. Unlike murine and human CMV, RCMV does not induce proteolytic degradation of MHC class I molecules. In RCMV-infected cells, the MHC class I molecules are stable, but their exit from the ER is delayed.
Assuntos
Regulação para Baixo , Antígenos de Histocompatibilidade Classe I/biossíntese , Muromegalovirus/fisiologia , Animais , Antígenos de Superfície/análise , Linhagem Celular , Células Cultivadas , Fibroblastos/virologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Transporte Proteico , Ratos , Fatores de TempoRESUMO
Relatively little is known about immune evasion during the productive phase of infection by the gamma(1)-herpesvirus Epstein-Barr virus (EBV). The use of a unique system to isolate cells in lytic cycle allowed us to identify a host shutoff function operating in productively EBV-infected B cells. This impairment of protein synthesis results from mRNA degradation induced upon expression of the early lytic-cycle gene product BGLF5. Recently, a gamma(2)-herpesvirus, Kaposi sarcoma herpesvirus, has also been shown to encode a host shutoff function, indicating that host shutoff appears to be a general feature of gamma-herpesviruses. One of the consequences of host shutoff is a block in the synthesis of HLA class I and II molecules, reflected by reduced levels of these antigen-presenting complexes at the surface of cells in EBV lytic cycle. This effect could lead to escape from T cell recognition and elimination of EBV-producing cells, thereby allowing generation of viral progeny in the face of memory T cell responses.
Assuntos
Linfócitos B/imunologia , Desoxirribonucleases/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Biossíntese de Proteínas/imunologia , Proteínas Virais/imunologia , Apresentação de Antígeno , Linfócitos B/citologia , Linhagem Celular Tumoral , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe II/biossíntese , Humanos , Microscopia de FluorescênciaRESUMO
Human adenovirus (HAdV) infection is a frequent and potentially severe complication following allogeneic stem cell transplantation in children. Because treatment with antiviral drugs is often ineffective, adoptive transfer of donor-derived HAdV-specific T cells able to control viral replication of HAdV of multiple serotypes may be an option for therapy. In healthy donors, predominantly HAdV-specific T cells expressing CD4 are detected. In this study, a preclinical in vitro model was used to measure the antiviral effect of HAdV-specific CD4+ T cells. CD4+ HAdV-specific T cell clones restricted by HLA class II molecules were generated and most of these clones recognized conserved peptides derived from the hexon protein. These cross-reactive T cell clones were able to control viral replication of multiple serotypes of HAdV in EBV-transformed B cells (B-LCL), melanoma cells (MJS) and primary bronchial epithelial cells through cognate interaction. The HAdV-specific CD4+ T cell clones were able to specifically lyse infected target cells using a perforin-dependent mechanism. Antigenic peptides were also presented to the CD4+ T cell clones when derived from endogenously produced hexon protein. Together, these results show that cross-reactive HAdV-specific CD4+ T cells can control replication of HAdV in vitro and provide a rationale for the use of HAdV-specific T cells in adoptive immunotherapy protocols for control of life-threatening HAdV-infections in immunocompromised patients.
Assuntos
Infecções por Adenovirus Humanos/terapia , Adenovírus Humanos/imunologia , Transferência Adotiva/métodos , Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Replicação Viral/imunologia , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Células Cultivadas , Células Clonais/imunologia , Reações Cruzadas/imunologia , Citotoxicidade Imunológica , HumanosRESUMO
Human herpesviruses, including EBV, persist for life in infected individuals. During the lytic replicative cycle that is required for the production of infectious virus and transmission to another host, many viral Ags are expressed. Especially at this stage, immune evasion strategies are likely to be advantageous to avoid elimination of virus-producing cells. However, little is known about immune escape during productive EBV infection because no fully permissive infection model is available. In this study, we have developed a novel strategy to isolate populations of cells in an EBV lytic cycle based on the expression of a reporter gene under the control of an EBV early lytic cycle promoter. Thus, induction of the viral lytic cycle in transfected EBV(+) B lymphoma cells resulted in concomitant reporter expression, allowing us, for the first time, to isolate highly purified cell populations in lytic cycle for biochemical and functional studies. Compared with latently infected B cells, cells supporting EBV lytic cycle displayed down-regulation of surface HLA class I, class II, and CD20, whereas expression levels of other surface markers remained unaffected. Moreover, during lytic cycle peptide transport into the endoplasmic reticulum, was reduced to <30% of levels found in latent infection. Because steady-state levels of TAP proteins were unaffected, these results point toward EBV-induced interference with TAP function as a specific mechanism contributing to the reduced levels of cell surface HLA class I. Our data implicate that EBV lytic cycle genes encode functions to evade T cell recognition, thereby creating a window for the generation of viral progeny.
Assuntos
Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Transportadores de Cassetes de Ligação de ATP , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/virologia , Linhagem Celular Tumoral , Separação Celular , Regulação para Baixo/imunologia , Infecções por Vírus Epstein-Barr/virologia , Genes Reporter , Antígenos HLA-D/biossíntese , Antígenos HLA-D/metabolismo , Herpesvirus Humano 4/genética , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/genética , Transporte Proteico/genética , Transporte Proteico/imunologia , Ratos , Proteínas Virais/biossíntese , Proteínas Virais/genética , Ativação Viral/genética , Ativação Viral/imunologia , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
Epstein-Barr virus (EBV) resides as a persistent infection in human leukocyte antigen (HLA) class II+ B lymphocytes and is associated with a number of malignancies. The EBV lytic-phase protein gp42 serves at least two functions: gp42 acts as the coreceptor for viral entry into B cells and hampers T-cell recognition via HLA class II molecules through steric hindrance of T-cell receptor-class II-peptide interactions. Here, we show that gp42 associates with class II molecules at their various stages of maturation, including immature alphabetaIi heterotrimers and mature alphabeta-peptide complexes. When analyzing the biosynthesis and maturation of gp42 in cells stably expressing the viral protein, we found that gp42 occurs in two forms: a full-length type II membrane protein and a truncated soluble form. Soluble gp42 is generated by proteolytic cleavage in the endoplasmic reticulum and is secreted. Soluble gp42 is sufficient to inhibit HLA class II-restricted antigen presentation to T cells. In an almost pure population of Burkitt's lymphoma cells in the EBV lytic cycle, both transmembrane and soluble forms of gp42 are detected. These results imply that soluble gp42 is generated during EBV lytic infection and could contribute to undetected virus production by mediating evasion from T-cell immunity.
Assuntos
Glicoproteínas/fisiologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Processamento de Proteína Pós-Traducional , Linfócitos T/imunologia , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Linfócitos B/virologia , Linhagem Celular Tumoral , Glicoproteínas/química , Humanos , Dados de Sequência Molecular , Proteínas Virais/químicaRESUMO
Detection and elimination of virus-infected cells by cytotoxic T lymphocytes depends on recognition of virus-derived peptides presented by MHC class I molecules. A critical step in this process is the translocation of peptides from the cytoplasm into the endoplasmic reticulum by the transporter associated with antigen processing (TAP). Here, we identified the bovine herpesvirus 1-encoded UL49.5 protein as a potent inhibitor of TAP. The expression of UL49.5 results in down-regulation of MHC class I molecules at the cell surface and inhibits detection and lysis of the cells by cytotoxic T lymphocytes. UL49.5 homologs encoded by two other varicelloviruses, pseudorabies-virus and equine herpesvirus 1, also block TAP. Homologs of UL49.5 are widely present in herpesviruses, acting as interaction partners for glycoprotein M, but in several varicelloviruses UL49.5 has uniquely evolved additional functions that mediate its participation in TAP inhibition. Inactivation of TAP by UL49.5 involves two events: inhibition of peptide transport through a conformational arrest of the transporter and degradation of TAP by proteasomes. UL49.5 is degraded along with TAP via a reaction that requires the cytoplasmic tail of UL49.5. Thus, UL49.5 represents a unique immune evasion protein that inactivates TAP through a unique two-tiered process.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Linfócitos T Citotóxicos/imunologia , Varicellovirus/imunologia , Varicellovirus/patogenicidade , Proteínas do Envelope Viral/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico Ativo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Varicellovirus/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/fisiologiaRESUMO
During co-evolution with its host, human cytomegalovirus has acquired multiple defense mechanisms to escape from immune recognition. In this study, we focused on US11, which binds to MHC class I heavy chains and mediates their dislocation to the cytosol and subsequent degradation by proteasomes. To examine which domains of class I heavy chains are involved in this process, we constructed chimeric HLA molecules of US11-sensitive and -insensitive class I molecules (HLA-A2 and HLA-G, respectively). Pulse-chase experiments were performed to evaluate protein stability and interactions between class I heavy chains and US11. Flow cytometry was employed to assess the effect of US11 on surface expression of the different chimeras. Our results indicate that the alpha1 and alpha2 domains of HLA molecules are important for the affinity of US11 association. However, the degradation efficiency seems to rely mostly on cytosolic tail residues. We found that the nonclassical HLA-G molecule is insensitive to US11-mediated degradation solely because it lacks essential tail residues. A deletion of the last two tail residues in full-length MHC class I molecules already caused a severe reduction in degradation efficiency. Altogether, our data provide new insights into the mechanism by which US11 down-regulates MHC class I molecules.
Assuntos
Regulação Viral da Expressão Gênica , Antígenos HLA/química , Antígeno HLA-A2/química , Antígenos de Histocompatibilidade Classe I/química , Proteínas de Ligação a RNA/fisiologia , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Sequência Consenso , Cisteína Endopeptidases/metabolismo , Citomegalovirus/fisiologia , Antígenos HLA/metabolismo , Antígeno HLA-A2/metabolismo , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Transfecção , Microglobulina beta-2/genéticaRESUMO
Human CMV (HCMV) can elude CTL as well as NK cells by modulating surface expression of MHC class I molecules. This strategy would be most efficient if the virus would selectively down-regulate viral Ag-presenting alleles, while at the same time preserving other alleles to act as inhibitors of NK cell activation. We focused on the HCMV unique short (US) region encoded protein US2, which binds to newly synthesized MHC class I H chains and supports their dislocation to the cytosol for subsequent degradation by proteasomes. We studied the effect of US2 on surface expression of individual class I locus products using flow cytometry. Our results were combined with crystal structure data of complexed US2/HLA-A2/beta(2)-microglobulin and alignments of 948 HLA class I database sequences of the endoplasmic reticulum lumenal region inplicated in US2 binding. This study suggests that surface expression of all HLA-A and -G and most HLA-B alleles will be affected by US2. Several HLA-B alleles and all HLA-C and -E alleles are likely to be insensitive to US2-mediated degradation. We also found that the MHC class I endoplasmic reticulum-lumenal domain alone is not sufficient for degradation by US2, as illustrated by the stability of soluble HLA-G1 in the presence of US2. Furthermore, we showed that the membrane-bound HLA-G1 isoform, but also tailless HLA-A2, are targeted for degradation. This indicates that the cytoplasmic tail of the MHC class I H chain is not required for its dislocation to the cytosol by US2.
Assuntos
Citomegalovirus/imunologia , Antígenos HLA/biossíntese , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/metabolismo , Glicoproteínas de Membrana/fisiologia , Proteínas Virais/fisiologia , Alelos , Sequência de Aminoácidos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/imunologia , Membrana Celular/metabolismo , Membrana Celular/virologia , Citomegalovirus/genética , Citoplasma/química , Citoplasma/genética , Citoplasma/imunologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Regulação da Expressão Gênica/imunologia , Marcadores Genéticos/imunologia , Antígenos HLA/química , Antígenos HLA/genética , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunidade Inata/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Ligação Proteica/genética , Ligação Proteica/imunologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Solubilidade , Transfecção , Proteínas do Envelope Viral , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Granulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Tyr704, Tyr729, Tyr744, Tyr764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significantly to G-CSF-induced proliferation, differentiation, and cell survival. However, it is unclear whether these tyrosines are equally important under more physiologic conditions. Here, we investigated how individual G-CSF-R tyrosines affect G-CSF responses of primary myeloid progenitors. We generated G-CSF-R-deficient mice and transduced their bone marrow cells with tyrosine "null" mutant (m0), single tyrosine "add-back" mutants, or wild-type (WT) receptors. G-CSF-induced responses were determined in primary colony assays, serial replatings, and suspension cultures. We show that removal of all tyrosines had no major influence on primary colony growth. However, adding back Tyr764 strongly enhanced proliferative responses, which was reverted by inhibition of ERK activity. Tyr729, which we found to be associated with the suppressor of cytokine signaling, SOCS3, had a negative effect on colony formation. After repetitive replatings, the clonogenic capacities of cells expressing m0 gradually dropped compared with WT. The presence of Tyr729, but also Tyr704 and Tyr744, both involved in activation of signal transducer and activator of transcription 3 (STAT3), further reduced replating efficiencies. Conversely, Tyr764 greatly elevated the clonogenic abilities of myeloid progenitors, resulting in a more than 10(4)-fold increase of colony-forming cells over m0 after the fifth replating. These findings suggest that tyrosines in the cytoplasmic domain of G-CSF-R, although dispensable for G-CSF-induced colony growth, recruit signaling mechanisms that regulate the maintenance and outgrowth of myeloid progenitor cells.
Assuntos
Fator Estimulador de Colônias de Granulócitos/farmacologia , Células Progenitoras Mieloides/citologia , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Proteínas Repressoras , Transdução de Sinais , Fatores de Transcrição , Tirosina/fisiologia , Animais , Células da Medula Óssea/metabolismo , Divisão Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Feminino , Masculino , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Mutação , Células Progenitoras Mieloides/metabolismo , Proteínas/metabolismo , Proteínas/fisiologia , Receptores de Fator Estimulador de Colônias de Granulócitos/fisiologia , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina , Transdução GenéticaRESUMO
Epstein-Barr virus (EBV) persists lifelong in infected hosts despite the presence of antiviral immunity. Many viral antigens are expressed during lytic infection. Thus, for EBV to spread, it must have evolved effective ways to evade immune recognition. Here, we report that HLA class II-restricted antigen presentation to T helper cells is hampered in the presence of the lytic-phase protein gp42. This interference with T cell activation involves association of gp42 with class II peptide complexes. Using HLA-DR tetramers, we identify a block in T cell receptor (TCR)-class II interactions imposed by gp42 as the underlying mechanism. EBV gp42 sterically clashes with TCR Valpha-domains as visualized by superimposing the crystal structures for gp42-HLA-DR1 and TCR-MHC class II complexes. Blocking TCR recognition provides a previously undescribed strategy for viral immune evasion.