RESUMEN
Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). Although ERAD components involved in degradation of luminal substrates are well characterized, much less is known about quality control of membrane proteins. Here, we analyzed the degradation pathways of two short-lived ER membrane model proteins in mammalian cells. Using a CRISPR-Cas9 genome-wide library screen, we identified an ERAD branch required for quality control of a subset of membrane proteins. Using biochemical and mass spectrometry approaches, we showed that this ERAD branch is defined by an ER membrane complex consisting of the ubiquitin ligase RNF185, the ubiquitin-like domain containing proteins TMUB1/2 and TMEM259/Membralin, a poorly characterized protein. This complex cooperates with cytosolic ubiquitin ligase UBE3C and p97 ATPase in degrading their membrane substrates. Our data reveal that ERAD branches have remarkable specificity for their membrane substrates, suggesting that multiple, perhaps combinatorial, determinants are involved in substrate selection.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Sistemas CRISPR-Cas , Línea Celular , Sistema Enzimático del Citocromo P-450/metabolismo , Células HEK293 , Células HeLa , Humanos , Dominios Proteicos , Pliegue de Proteína , Proteolisis , Proteínas de Saccharomyces cerevisiae/metabolismo , Esterol 14-Desmetilasa/metabolismoRESUMEN
The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHHMHCII-SpikeRBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.
Asunto(s)
Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/farmacología , COVID-19/inmunología , COVID-19/prevención & control , Pandemias , SARS-CoV-2/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , COVID-19/epidemiología , Vacunas contra la COVID-19/administración & dosificación , Camélidos del Nuevo Mundo/inmunología , Femenino , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunización Secundaria , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Pandemias/prevención & control , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , SARS-CoV-2/genética , Anticuerpos de Dominio Único/administración & dosificación , Anticuerpos de Dominio Único/inmunología , Glicoproteína de la Espiga del Coronavirus/administración & dosificación , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Bovine herpesvirus 1 (BoHV-1)-encoded UL49.5 (a homologue of herpesvirus glycoprotein N) can combine different functions, regulated by complex formation with viral glycoprotein M (gM). We aimed to identify the mechanisms governing the immunomodulatory activity of BoHV-1 UL49.5. In this study, we addressed the impact of gM/UL49.5-specific regions on heterodimer formation, folding and trafficking from the endoplasmic reticulum (ER) to the trans-Golgi network (TGN) - events previously found to be responsible for abrogation of the UL49.5-mediated inhibition of the transporter associated with antigen processing (TAP). We first established, using viral mutants, that no other viral protein could efficiently compensate for the chaperone function of UL49.5 within the complex. The cytoplasmic tail of gM, containing putative trafficking signals, was dispensable either for ER retention of gM or for the release of the complex. We constructed cell lines with stable co-expression of BoHV-1 gM with chimeric UL49.5 variants, composed of the BoHV-1 N-terminal domain fused to the transmembrane region (TM) from UL49.5 of varicella-zoster virus or TM and the cytoplasmic tail of influenza virus haemagglutinin. Those membrane-anchored N-terminal domains of UL49.5 were sufficient to form a complex, yet gM/UL49.5 folding and ER-TGN trafficking could be affected by the UL49.5 TM sequence. Finally, we found that leucine substitutions in putative glycine zipper motifs within TM helices of gM resulted in strong reduction of complex formation and decreased ability of gM to interfere with UL49.5-mediated major histocompatibility class I downregulation. These findings highlight the importance of gM/UL49.5 transmembrane domains for the biology of this conserved herpesvirus protein complex.
Asunto(s)
Enfermedades de los Bovinos/virología , Retículo Endoplásmico/virología , Aparato de Golgi/virología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Bovino 1/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Bovinos , Infecciones por Herpesviridae/virología , Herpesvirus Bovino 1/química , Herpesvirus Bovino 1/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
Misfolded endoplasmic reticulum (ER) proteins are dislocated towards the cytosol and degraded by the ubiquitin-proteasome system in a process called ER-associated protein degradation (ERAD). During infection with human cytomegalovirus (HCMV), the viral US2 protein targets HLA class I molecules (HLA-I) for degradation via ERAD to avoid elimination by the immune system. US2-mediated degradation of HLA-I serves as a paradigm of ERAD and has facilitated the identification of TRC8 (also known as RNF139) as an E3 ubiquitin ligase. No specific E2 enzymes had previously been described for cooperation with TRC8. In this study, we used a lentiviral CRISPR/Cas9 library targeting all known human E2 enzymes to assess their involvement in US2-mediated HLA-I downregulation. We identified multiple E2 enzymes involved in this process, of which UBE2G2 was crucial for the degradation of various immunoreceptors. UBE2J2, on the other hand, counteracted US2-induced ERAD by downregulating TRC8 expression. These findings indicate the complexity of cellular quality control mechanisms, which are elegantly exploited by HCMV to elude the immune system.
Asunto(s)
Citomegalovirus/metabolismo , Regulación hacia Abajo , Receptores Inmunológicos/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Sistemas CRISPR-Cas/genética , Pruebas Genéticas , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Modelos Biológicos , Proteolisis , Receptores de Superficie Celular/metabolismo , Células U937 , Regulación hacia ArribaRESUMEN
Type I IFNs play critical roles in orchestrating the antiviral defense by inducing direct antiviral activities and shaping the adaptive immune response. Viruses have evolved numerous strategies to specifically interfere with IFN production or its downstream mediators, thereby allowing successful infection of the host to occur. The prototypic human gammaherpesvirus EBV, which is associated with infectious mononucleosis and malignant tumors, harbors many immune-evasion proteins that manipulate the adaptive and innate immune systems. In addition to proteins, the virus encodes >40 mature microRNAs for which the functions remain largely unknown. In this article, we identify EBV-encoded miR-BART16 as a novel viral immune-evasion factor that interferes with the type I IFN signaling pathway. miR-BART16 directly targets CREB-binding protein, a key transcriptional coactivator in IFN signaling, thereby inducing CREB-binding protein downregulation in EBV-transformed B cells and gastric carcinoma cells. miR-BART16 abrogates the production of IFN-stimulated genes in response to IFN-α stimulation and it inhibits the antiproliferative effect of IFN-α on latently infected BL cells. By obstructing the type I IFN-induced antiviral response, miR-BART16 provides a means to facilitate the establishment of latent EBV infection and enhance viral replication.
Asunto(s)
Herpesvirus Humano 4/genética , Interferón Tipo I/metabolismo , MicroARNs/metabolismo , ARN Viral/metabolismo , Transducción de Señal , Proteína de Unión a CREB/metabolismo , Línea Celular , Herpesvirus Humano 4/inmunología , Interacciones Huésped-Patógeno , Humanos , Evasión Inmune , Inmunidad Innata , Interferón Tipo I/inmunología , MicroARNs/genética , ARN Viral/genética , Replicación ViralRESUMEN
The MHC class I antigen presentation pathway enables cells infected with intracellular pathogens to signal the presence of the invader to the immune system. Cytotoxic T lymphocytes are able to eliminate the infected cells through recognition of pathogen-derived peptides presented by MHC class I molecules at the cell surface. In the course of evolution, many viruses have acquired inhibitors that target essential stages of the MHC class I antigen presentation pathway. Studies on these immune evasion proteins reveal fascinating strategies used by viruses to elude the immune system. Viral immunoevasins also constitute great research tools that facilitate functional studies on the MHC class I antigen presentation pathway, allowing the investigation of less well understood routes, such as TAP-independent antigen presentation and cross-presentation of exogenous proteins. Viral immunoevasins have also helped to unravel more general cellular processes. For instance, basic principles of ER-associated protein degradation via the ubiquitin-proteasome pathway have been resolved using virus-induced degradation of MHC class I as a model. This review highlights how viral immunoevasins have increased our understanding of MHC class I-restricted antigen presentation.
Asunto(s)
Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/inmunología , Evasión Inmune , Animales , Retículo Endoplásmico/metabolismo , Humanos , Péptidos/inmunología , Virus/inmunologíaRESUMEN
Poxviruses comprise a group of large dsDNA viruses that include members relevant to human and animal health, such as variola virus, monkeypox virus, cowpox virus and vaccinia virus (VACV). Poxviruses are remarkable for their unique replication cycle, which is restricted to the cytoplasm of infected cells. The independence from the host nucleus requires poxviruses to encode most of the enzymes involved in DNA replication, transcription and processing. Here, we use the CRISPR/Cas9 genome engineering system to induce DNA damage to VACV (strain Western Reserve) genomes. We show that targeting CRISPR/Cas9 to essential viral genes limits virus replication efficiently. Although VACV is a strictly cytoplasmic pathogen, we observed extensive viral genome editing at the target site; this is reminiscent of a non-homologous end-joining DNA repair mechanism. This pathway was not dependent on the viral DNA ligase, but critically involved the cellular DNA ligase IV. Our data show that DNA ligase IV can act outside of the nucleus to allow repair of dsDNA breaks in poxvirus genomes. This pathway might contribute to the introduction of mutations within the genome of poxviruses and may thereby promote the evolution of these viruses.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN Ligasa (ATP)/metabolismo , Reparación del ADN , Genoma Viral , Interacciones Microbiota-Huesped/genética , Virus Vaccinia/genética , Sistemas CRISPR-Cas , Línea Celular Tumoral , Citosol/metabolismo , Citosol/virología , ADN Ligasa (ATP)/genética , Replicación del ADN , ADN Viral/genética , Células HEK293 , Humanos , Mutagénesis , Virus Vaccinia/fisiología , Replicación Viral/genéticaRESUMEN
Herpesviruses infect the majority of the human population and can cause significant morbidity and mortality. Herpes simplex virus (HSV) type 1 causes cold sores and herpes simplex keratitis, whereas HSV-2 is responsible for genital herpes. Human cytomegalovirus (HCMV) is the most common viral cause of congenital defects and is responsible for serious disease in immuno-compromised individuals. Epstein-Barr virus (EBV) is associated with infectious mononucleosis and a broad range of malignancies, including Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, and post-transplant lymphomas. Herpesviruses persist in their host for life by establishing a latent infection that is interrupted by periodic reactivation events during which replication occurs. Current antiviral drug treatments target the clinical manifestations of this productive stage, but they are ineffective at eliminating these viruses from the infected host. Here, we set out to combat both productive and latent herpesvirus infections by exploiting the CRISPR/Cas9 system to target viral genetic elements important for virus fitness. We show effective abrogation of HCMV and HSV-1 replication by targeting gRNAs to essential viral genes. Simultaneous targeting of HSV-1 with multiple gRNAs completely abolished the production of infectious particles from human cells. Using the same approach, EBV can be almost completely cleared from latently infected EBV-transformed human tumor cells. Our studies indicate that the CRISPR/Cas9 system can be effectively targeted to herpesvirus genomes as a potent prophylactic and therapeutic anti-viral strategy that may be used to impair viral replication and clear latent virus infection.
Asunto(s)
Sistemas CRISPR-Cas/genética , Citomegalovirus/genética , Edición Génica/métodos , Genoma Viral , Infecciones por Herpesviridae/genética , Herpesviridae/genética , Línea Celular , Herpesvirus Humano 1 , Humanos , Reacción en Cadena de la Polimerasa , Latencia del Virus/genéticaRESUMEN
Cell-mediated immunity plays a key role in host control of viral infection. This is exemplified by life-threatening reactivations of e.g. herpesviruses in individuals with impaired T-cell and/or iNKT cell responses. To allow lifelong persistence and virus production in the face of primed immunity, herpesviruses exploit immune evasion strategies. These include a reduction in viral antigen expression during latency and a number of escape mechanisms that target antigen presentation pathways. Given the plethora of foreign antigens expressed in virus-producing cells, herpesviruses are conceivably most vulnerable to elimination by cell-mediated immunity during the replicative phase of infection. Here, we show that a prototypic herpesvirus, Epstein-Barr virus (EBV), encodes a novel, broadly acting immunoevasin, gp150, that is expressed during the late phase of viral replication. In particular, EBV gp150 inhibits antigen presentation by HLA class I, HLA class II, and the non-classical, lipid-presenting CD1d molecules. The mechanism of gp150-mediated T-cell escape does not depend on degradation of the antigen-presenting molecules nor does it require gp150's cytoplasmic tail. Through its abundant glycosylation, gp150 creates a shield that impedes surface presentation of antigen. This is an unprecedented immune evasion mechanism for herpesviruses. In view of its likely broader target range, gp150 could additionally have an impact beyond escape of T cell activation. Importantly, B cells infected with a gp150-null mutant EBV displayed rescued levels of surface antigen presentation by HLA class I, HLA class II, and CD1d, supporting an important role for iNKT cells next to classical T cells in fighting EBV infection. At the same time, our results indicate that EBV gp150 prolongs the timespan for producing viral offspring at the most vulnerable stage of the viral life cycle.
Asunto(s)
Presentación de Antígeno/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Evasión Inmune/inmunología , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas Virales/inmunología , Western Blotting , Citometría de Flujo , Herpesvirus Humano 4/inmunología , Humanos , Microscopía Confocal , Linfocitos T/inmunología , Transducción GenéticaRESUMEN
MicroRNAs (miRNAs) are small RNA molecules that post-transcriptionally regulate gene expression through silencing of complementary target mRNAs. miRNAs are involved in many biological processes, including cell proliferation, differentiation, cell signaling and cellular defense responses to infection. Strategies that allow for strong and stable suppression of specific microRNA activity are needed to study miRNA functions and to develop therapeutic intervention strategies aimed at interfering with miRNA activity in vivo. One of these classes of miRNA inhibitors are Tough Decoys (TuD) RNAs, which comprise of an imperfect RNA hairpin structure that harbors two opposing miRNA binding sites. Upon developing TuDs targeting Epstein-Barr virus miRNAs, we observed a strong variation in inhibitory potential between different TuD RNAs targeting the same miRNA. We show that the composition of the 'bulge' sequence in the miRNA binding sites has a strong impact on the inhibitory potency of the TuD. Our data implies that miRNA inhibition correlates with the thermodynamic properties of the TuD and that design aimed at lowering the TuD opening energy increases TuD potency. Our study provides specific guidelines for the design and construction of potent decoy-based miRNA inhibitors, which may be used for future therapeutic intervention strategies.
Asunto(s)
MicroARNs/genética , Conformación de Ácido Nucleico , ARN/genética , Sitios de Unión , Herpesvirus Humano 4/química , Herpesvirus Humano 4/genética , Humanos , MicroARNs/antagonistas & inhibidores , MicroARNs/química , ARN/química , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , TermodinámicaRESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/inmunología , Evasión Inmune/inmunología , Proteínas Virales/inmunología , Virosis/inmunología , Animales , Evolución Biológica , Humanos , Evasión Inmune/genética , Filogenia , Virosis/genética , Latencia del Virus/inmunologíaRESUMEN
BACKGROUND: Epstein-Barr virus (EBV) establishes lifelong infections in its human host. The virus is associated with a broad range of malignancies of lymphoid and epithelial origin, including Burkitt's lymphoma, post-transplant lymphoproliferative disease, nasopharyngeal carcinoma and gastric carcinoma. During the latent phase of its life cycle, EBV expresses more than 40 mature miRNAs that are highly abundant in tumor cells and may contribute to oncogenesis. Although multiple studies have assessed the relative expression profiles of EBV miRNAs in tumor cells, data linking these expression levels to functional target knockdown are mostly lacking. Therefore we set out to systematically assess the EBV miRNA expression levels in EBV(+) tumor cell lines, and correlate this to their functional silencing capacity in these cells. RESULTS: We provide comprehensive EBV miRNA expression profiles of the EBV(+) cell lines C666-1 (nasopharyngeal carcinoma), SNU-719 (gastric carcinoma), Jijoye (Burkitt's lymphoma), and AKBM (Burkitt's lymphoma) and of EBV(-) cells ectopically expressing the BART miRNA cluster. By deep sequencing the small RNA population and conducting miRNA-reporter experiments to assay miRNA potency, we were able to compare the expression profiles of the EBV miRNAs with their functional silencing efficacy. We observe a strong correlation between miRNA expression levels and functional miRNA activity. There is large variation in expression levels between EBV miRNAs in a given cell line, whereas the relative expression profiles are well maintained between cell lines. Furthermore, we show that miRNA arm selection bias is less pronounced for gamma-herpesvirus miRNAs than for human miRNAs. CONCLUSION: We provide an in depth assessment of the expression levels and silencing activity of all EBV miRNAs in B- and epithelial cell lines of different latency stages. Our data show a good correlation between relative EBV miRNA expression levels and silencing capacity, and suggest preferential processing of particular EBV miRNAs irrespective of cell-type. In addition to encoding the largest number of precursor miRNAs of all human herpesviruses, EBV expresses many miRNAs precursors that yield two functional miRNA strands, rather than one guide strand and a non-functional passenger strand. This reduced strand bias may increase the size of the EBV miRNA targetome.
Asunto(s)
Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/genética , MicroARNs/genética , ARN Viral/genética , Transcriptoma , Línea Celular Tumoral , Expresión Génica , Genes Reporteros , Secuenciación de Nucleótidos de Alto Rendimiento , HumanosRESUMEN
Epstein-Bar virus (EBV) is widespread within the human population with over 90% of adults being infected. In response to primary EBV infection, the host mounts an antiviral immune response comprising both innate and adaptive effector functions. Although the immune system can control EBV infection to a large extent, the virus is not cleared. Instead, EBV establishes a latent infection in B lymphocytes characterized by limited viral gene expression. For the production of new viral progeny, EBV reactivates from these latently infected cells. During the productive phase of infection, a repertoire of over 80 EBV gene products is expressed, presenting a vast number of viral antigens to the primed immune system. In particular the EBV-specific CD4+ and CD8+ memory T lymphocytes can respond within hours, potentially destroying the virus-producing cells before viral replication is completed and viral particles have been released. Preceding the adaptive immune response, potent innate immune mechanisms provide a first line of defense during primary and recurrent infections. In spite of this broad range of antiviral immune effector mechanisms, EBV persists for life and continues to replicate. Studies performed over the past decades have revealed a wide array of viral gene products interfering with both innate and adaptive immunity. These include EBV-encoded proteins as well as small noncoding RNAs with immune-evasive properties. The current review presents an overview of the evasion strategies that are employed by EBV to facilitate immune escape during latency and productive infection. These evasion mechanisms may also compromise the elimination of EBV-transformed cells, and thus contribute to malignancies associated with EBV infection.
Asunto(s)
Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Evasión Inmune , Animales , Infecciones por Virus de Epstein-Barr/virología , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/genética , HumanosRESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Virus de la Viruela Vacuna/inmunología , Evasión Inmune/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas Virales/inmunología , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Presentación de Antígeno/genética , Presentación de Antígeno/inmunología , Línea Celular Tumoral , Membrana Celular/metabolismo , Virus de la Viruela Vacuna/genética , Retículo Endoplásmico/inmunología , Mutación del Sistema de Lectura , Células HEK293 , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Unión Proteica/inmunología , Transporte de Proteínas/inmunología , Proteínas Virales/genéticaRESUMEN
Genomic variation and related evolutionary dynamics of human respiratory syncytial virus (RSV), a common causative agent of severe lower respiratory tract infections, may affect its transmission behavior. RSV evolutionary patterns are likely to be influenced by a precarious interplay between selection favoring variants with higher replicative fitness and variants that evade host immune responses. Studying RSV genetic variation can reveal both the genes and the individual codons within these genes that are most crucial for RSV survival. In this study, we conducted genetic diversity and evolutionary rate analyses on 36 RSV subgroup B (RSV-B) whole-genome sequences. The attachment protein, G, was the most variable protein; accordingly, the G gene had a higher substitution rate than other RSV-B genes. Overall, less genetic variability was found among the available RSV-B genome sequences than among RSV-A genome sequences in a comparable sample. The mean substitution rates of the two subgroups were, however, similar (for subgroup A, 6.47 × 10(-4) substitutions/site/year [95% credible interval {CI 95%}, 5.56 × 10(-4) to 7.38 × 10(-4)]; for subgroup B, 7.76 × 10(-4) substitutions/site/year [CI 95%, 6.89 × 10(-4) to 8.58 × 10(-4)]), with the time to their most recent common ancestors (TMRCAs) being much lower for RSV-B (19 years) than for RSV-A (46.8 years). The more recent RSV-B TMRCA is apparently the result of a genetic bottleneck that, over longer time scales, is still compatible with neutral population dynamics. Whereas the immunogenic G protein seems to require high substitution rates to ensure immune evasion, strong purifying selection in conserved proteins such as the fusion protein and nucleocapsid protein is likely essential to preserve RSV viability.
Asunto(s)
Evolución Molecular , Variación Genética/genética , Genómica/métodos , Filogenia , Virus Sincitial Respiratorio Humano/genética , Secuencia de Aminoácidos , Secuencia de Bases , Teorema de Bayes , Evasión Inmune/genética , Funciones de Verosimilitud , Modelos Genéticos , Datos de Secuencia Molecular , Dinámica Poblacional , Selección Genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Especificidad de la Especie , Replicación Viral/genéticaRESUMEN
CD4(+) T cells play a central role in adaptive immunity. The acknowledgment of their cytolytic effector function and the finding that endogenous antigens can enter the HLA class II processing pathway make CD4(+) T cells promising tools for immunotherapy. Expression of HLA class II and endogenous antigen, however, does not always correlate with T-cell recognition. We therefore investigated processing and presentation of endogenous HLA class II epitopes that induced CD4(+) T cells during in vivo immune responses. We demonstrate that the peptide editor HLA-DM allowed antigen presentation of some (DM-resistant antigens) but abolished surface expression of other natural HLA class II epitopes (DM-sensitive antigens). DM sensitivity was shown to be epitope specific, mediated via interaction between HLA-DM and the HLA-DR restriction molecule, and reversible by HLA-DO. Because of the restricted expression of HLA-DO, presentation of DM-sensitive antigens was limited to professional antigen-presenting cells, whereas DM-resistant epitopes were expressed on all HLA class II-expressing cells. In conclusion, our data provide novel insights into the presentation of endogenous HLA class II epitopes and identify intracellular antigen processing and presentation as a critical factor for CD4(+) T-cell recognition. This opens perspectives to exploit selective processing capacities as a new approach for targeted immunotherapy.
Asunto(s)
Presentación de Antígeno/inmunología , Linfocitos T CD4-Positivos/inmunología , Epítopos/inmunología , Antígenos HLA-D/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Células Presentadoras de Antígenos/inmunología , Biomarcadores/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Antígenos HLA-D/química , Antígenos HLA-D/metabolismo , Antígenos HLA-D/farmacología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fragmentos de Péptidos/inmunología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Type 1 diabetes (T1D) is characterized by HLA class I-mediated presentation of autoantigens on the surface of pancreatic ß-cells. Recognition of these autoantigens by CD8+ T cells results in the destruction of pancreatic ß-cells and, consequently, insulin deficiency. Most epitopes presented at the surface of ß-cells derive from the insulin precursor molecule proinsulin. The intracellular processing pathway(s) involved in the generation of these peptides are poorly defined. In this study, we show that a proinsulin B-chain antigen (PPIB5-14) originates from proinsulin molecules that are processed by ER-associated protein degradation (ERAD) and thus originate from ER-resident proteins. Furthermore, screening genes encoding for E2 ubiquitin conjugating enzymes, we identified UBE2G2 to be involved in proinsulin degradation and subsequent presentation of the PPIB10-18 autoantigen. These insights into the pathway involved in the generation of insulin-derived peptides emphasize the importance of proinsulin processing in the ER to T1D pathogenesis and identify novel targets for future T1D therapies.
Asunto(s)
Autoantígenos , Degradación Asociada con el Retículo Endoplásmico , Proinsulina , Proteolisis , Enzimas Ubiquitina-Conjugadoras , Proinsulina/metabolismo , Proinsulina/inmunología , Proinsulina/genética , Autoantígenos/metabolismo , Autoantígenos/inmunología , Humanos , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Presentación de Antígeno/inmunología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/inmunologíaRESUMEN
Herpes simplex virus type 1 (HSV-1) is a neurotropic alphaherpesvirus that establishes a lifelong infection in sensory neurons of infected individuals, accompanied with intermittent reactivation of latent virus causing (a)symptomatic virus shedding. Whereas acyclovir (ACV) is a safe and highly effective antiviral to treat HSV-1 infections, long-term usage can lead to emergence of ACV resistant (ACVR) HSV-1 and subsequently ACV refractory disease. Here, we isolated an HSV-1 strain from a patient with reactivated herpetic eye disease that did not respond to ACV treatment. The isolate carried a novel non-synonymous F289S mutation in the viral UL23 gene encoding the thymidine kinase (TK) protein. Because ACV needs conversion by viral TK and subsequently cellular kinases to inhibit HSV-1 replication, the UL23 gene is commonly mutated in ACVR HSV-1 strains. The potential role of the F289S mutation causing ACVR was investigated using CRISPR/Cas9-mediated HSV-1 genome editing. Reverting the F289S mutation in the original clinical isolate to the wild-type sequence S289F resulted in an ACV-sensitive (ACVS) phenotype, and introduction of the F289S substitution in an ACVS HSV-1 reference strain led to an ACVR phenotype. In summary, we identified a new HSV-1 TK mutation in the eye of a patient with ACV refractory herpetic eye disease, which was identified as the causative ACVR mutation with the aid of CRISPR/Cas9-mediated genome engineering technology. Direct editing of clinical HSV-1 isolates by CRISPR/Cas9 is a powerful strategy to assess whether single residue substitutions are causative to a clinical ACVR phenotype.
Asunto(s)
Aciclovir , Antivirales , Sistemas CRISPR-Cas , Farmacorresistencia Viral , Edición Génica , Herpesvirus Humano 1 , Mutación , Timidina Quinasa , Timidina Quinasa/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/enzimología , Humanos , Farmacorresistencia Viral/genética , Aciclovir/farmacología , Aciclovir/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , Herpes Simple/virología , Herpes Simple/tratamiento farmacológicoRESUMEN
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.