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1.
PLoS One ; 19(5): e0287877, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38787820

RESUMEN

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/inmunología
2.
Proc Natl Acad Sci U S A ; 118(44)2021 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-34654739

RESUMEN

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/inmunología
4.
Cells ; 9(9)2020 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-32872420

RESUMEN

The ongoing threat of viral infections and the emergence of antiviral drug resistance warrants a ceaseless search for new antiviral compounds. Broadly-inhibiting compounds that act on elements shared by many viruses are promising antiviral candidates. Here, we identify a peptide derived from the cowpox virus protein CPXV012 as a broad-spectrum antiviral peptide. We found that CPXV012 peptide hampers infection by a multitude of clinically and economically important enveloped viruses, including poxviruses, herpes simplex virus-1, hepatitis B virus, HIV-1, and Rift Valley fever virus. Infections with non-enveloped viruses such as Coxsackie B3 virus and adenovirus are not affected. The results furthermore suggest that viral particles are neutralized by direct interactions with CPXV012 peptide and that this cationic peptide may specifically bind to and disrupt membranes composed of the anionic phospholipid phosphatidylserine, an important component of many viral membranes. The combined results strongly suggest that CPXV012 peptide inhibits virus infections by direct interactions with phosphatidylserine in the viral envelope. These results reiterate the potential of cationic peptides as broadly-acting virus inhibitors.


Asunto(s)
Antivirales/uso terapéutico , Péptidos/metabolismo , Fosfatidilserinas/metabolismo , Envoltura Viral/metabolismo , Antivirales/farmacología , Humanos
5.
Mol Cell ; 79(5): 768-781.e7, 2020 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-32738194

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/metabolismo
6.
Cells ; 8(12)2019 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-31817841

RESUMEN

Transporter associated with antigen processing (TAP), a key player in the major histocompatibility complex class I-restricted antigen presentation, makes an attractive target for viruses that aim to escape the immune system. Mechanisms of TAP inhibition vary among virus species. Bovine herpesvirus 1 (BoHV-1) is unique in its ability to target TAP for proteasomal degradation following conformational arrest by the UL49.5 gene product. The exact mechanism of TAP removal still requires elucidation. For this purpose, a TAP-GFP (green fluorescent protein) fusion protein is instrumental, yet GFP-tagging may affect UL49.5-induced degradation. Therefore, we constructed a series of TAP-GFP variants using various linkers to obtain an optimal cellular fluorescent TAP platform. Mel JuSo (MJS) cells with CRISPR/Cas9 TAP1 or TAP2 knockouts were reconstituted with TAP-GFP constructs. Our results point towards a critical role of GFP localization on fluorescent properties of the fusion proteins and, in concert with the type of a linker, on the susceptibility to virally-induced inhibition and degradation. The fluorescent TAP platform was also used to re-evaluate TAP stability in the presence of other known viral TAP inhibitors, among which only UL49.5 was able to reduce TAP levels. Finally, we provide evidence that BoHV-1 UL49.5-induced TAP removal is p97-dependent, which indicates its degradation via endoplasmic reticulum-associated degradation (ERAD).


Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2/metabolismo , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP/metabolismo , Herpesvirus Bovino 1/patogenicidad , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2/antagonistas & inhibidores , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP/antagonistas & inhibidores , Acetanilidas/farmacología , Animales , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/genética , Benzotiazoles/farmacología , Bovinos , Línea Celular , Línea Celular Tumoral , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Immunoblotting , Inmunoprecipitación , Plásmidos/genética
7.
Methods Mol Biol ; 1988: 187-198, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31147941

RESUMEN

MHC class I molecules are an important component of the cell-mediated immune defense, presenting peptides to surveilling CD8+ cytotoxic T cells. During viral infection, MHC class I molecules carry and display viral peptides at the cell surface. CD8+ T cells that recognize these peptides will eliminate the virus-infected cells. Viruses counteract this highly sophisticated host detection system by downregulating cell surface expression of MHC class I molecules.In this chapter, we describe a flow cytometry-based method that can be used for the identification of viral gene products potentially responsible for evasion from MHC class I-restricted antigen presentation. The gene(s) of interest are expressed constitutively through lentiviral transduction of cells. Subsequently, MHC I surface expression is monitored using MHC class I-specific antibodies. Once the viral gene product responsible for MHC I downregulation has been identified, the same cells can be used to elucidate the mechanism of action. The stage at which interference with antigen processing occurs can be identified using specific assays. An essential step frequently targeted by viruses is the translocation of peptides into the ER by the transporter associated with antigen processing, TAP. TAP function can be measured using a highly specific in vitro assay involving flow cytometric evaluation of the import of a fluorescent peptide substrate.The protocol described in this chapter enables the identification of virus-encoded MHC class I inhibitors that hinder antigen processing and presentation. Subsequently, their mechanism of action can be unraveled; this knowledge may help to rectify their actions.


Asunto(s)
Presentación de Antígeno/inmunología , Citometría de Flujo/métodos , Antígenos de Histocompatibilidad Clase I/inmunología , Virus/inmunología , Transportadoras de Casetes de Unión a ATP/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Regulación hacia Abajo , Humanos , Lentivirus/metabolismo , Péptidos/metabolismo , Polietileneimina/química , Transducción Genética
8.
J Gen Virol ; 100(3): 497-510, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30694168

RESUMEN

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/genética
9.
Mol Immunol ; 113: 103-114, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-29606337

RESUMEN

Several hundred million years of co-evolution of vertebrates and invading pathogens have shaped the adaptive immune system to fight back the unwanted invaders through highly sophisticated defense mechanisms. Herpesviruses manage to dodge this immune response by hampering one of the central hinges of human adaptive immunity, the major histocompatibility complex (MHC) class I antigen presentation pathway. One of the bottlenecks of this pathway is the loading of pathogen-derived peptides onto MHC-I molecules in the endoplasmic reticulum (ER). This task is accomplished by the MHC class I peptide-loading complex (PLC), of which the transporter associated with antigen-processing (TAP) is a central component. In this review, we summarize recent structural and functional insights into the molecular architecture of the PLC, how TAP accomplishes the transport of peptides across the ER membrane, and how herpes- and poxviruses inhibit TAP-mediated peptide translocation and subsequent antigen presentation.


Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Evasión Inmune/inmunología , Péptidos/inmunología , Inmunidad Adaptativa/inmunología , Animales , Retículo Endoplásmico/inmunología , Humanos
10.
RNA Biol ; 15(11): 1410-1419, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30339041

RESUMEN

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 , Termodinámica
11.
J Gen Virol ; 99(6): 790-804, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29676720

RESUMEN

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/genética
12.
J Cell Sci ; 130(17): 2883-2892, 2017 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-28743740

RESUMEN

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 Arriba
13.
J Immunol ; 198(10): 4062-4073, 2017 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-28416598

RESUMEN

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 Viral
14.
Sci Rep ; 7: 41968, 2017 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-28176813

RESUMEN

HIV presents one of the highest evolutionary rates ever detected and combination antiretroviral therapy is needed to overcome the plasticity of the virus population and control viral replication. Conventional treatments lack the ability to clear the latent reservoir, which remains the major obstacle towards a cure. Novel strategies, such as CRISPR/Cas9 gRNA-based genome-editing, can permanently disrupt the HIV genome. However, HIV genome-editing may accelerate viral escape, questioning the feasibility of the approach. Here, we demonstrate that CRISPR/Cas9 targeting of single HIV loci, only partially inhibits HIV replication and facilitates rapid viral escape at the target site. A combinatorial approach of two strong gRNAs targeting different regions of the HIV genome can completely abrogate viral replication and prevent viral escape. Our data shows that the accelerating effect of gene-editing on viral escape can be overcome and as such gene-editing may provide a future alternative for control of HIV-infection.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Genoma Viral , Infecciones por VIH/terapia , VIH-1/genética , ARN Guía de Kinetoplastida/genética , Replicación Viral/genética , Marcación de Gen , Células HEK293 , Infecciones por VIH/virología , Humanos , Células Jurkat
15.
Viruses ; 8(11)2016 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-27854284

RESUMEN

Misfolded proteins from the endoplasmic reticulum (ER) are transported back into the cytosol for degradation via the ubiquitin-proteasome system. The human cytomegalovirus protein US11 hijacks this ER-associated protein degradation (ERAD) pathway to downregulate human leukocyte antigen (HLA) class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. Recently, we identified the E3 ubiquitin ligase transmembrane protein 129 (TMEM129) as a key player in this process, where interference with TMEM129 activity in human cells completely abrogates US11-mediated class I degradation. Here, we set out to further characterize TMEM129. We show that TMEM129 is a non-glycosylated protein containing a non-cleaved signal anchor sequence. By glycosylation scanning mutagenesis, we show that TMEM129 is a tri-spanning ER-membrane protein that adopts an Nexo-Ccyto orientation. This insertion in the ER membrane positions the C-terminal really interesting new gene (RING) domain of TMEM129 in the cytosol, making it available to catalyze ubiquitination reactions that are required for cytosolic degradation of secretory proteins.


Asunto(s)
Retículo Endoplásmico/química , Membranas Intracelulares/química , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Ubiquitina-Proteína Ligasas/análisis , Ubiquitina-Proteína Ligasas/metabolismo , Humanos , Modelos Biológicos , Ubiquitinación
16.
BMC Genomics ; 17: 644, 2016 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-27531524

RESUMEN

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 , Humanos
17.
PLoS Pathog ; 12(6): e1005701, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27362483

RESUMEN

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/genética
19.
PLoS Pathog ; 12(4): e1005550, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-27077376

RESUMEN

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 Genética
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