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1.
Nature ; 600(7890): 701-706, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34673755

RESUMEN

Following severe adverse reactions to the AstraZeneca ChAdOx1-S-nCoV-19 vaccine1,2, European health authorities recommended that patients under the age of 55 years who received one dose of ChAdOx1-S-nCoV-19 receive a second dose of the Pfizer BNT162b2 vaccine as a booster. However, the effectiveness and the immunogenicity of this vaccination regimen have not been formally tested. Here we show that the heterologous ChAdOx1-S-nCoV-19 and BNT162b2 combination confers better protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection than the homologous BNT162b2 and BNT162b2 combination in a real-world observational study of healthcare workers (n = 13,121). To understand the underlying mechanism, we conducted a longitudinal survey of the anti-spike immunity conferred by each vaccine combination. Both combinations induced strong anti-spike antibody responses, but sera from heterologous vaccinated individuals displayed a stronger neutralizing activity regardless of the SARS-CoV-2 variant. This enhanced neutralizing potential correlated with increased frequencies of switched and activated memory B cells that recognize the SARS-CoV-2 receptor binding domain. The ChAdOx1-S-nCoV-19 vaccine induced a weaker IgG response but a stronger T cell response than the BNT162b2 vaccine after the priming dose, which could explain the complementarity of both vaccines when used in combination. The heterologous vaccination regimen could therefore be particularly suitable for immunocompromised individuals.


Asunto(s)
Vacuna BNT162/administración & dosificación , Vacuna BNT162/inmunología , COVID-19/inmunología , COVID-19/prevención & control , ChAdOx1 nCoV-19/administración & dosificación , ChAdOx1 nCoV-19/inmunología , SARS-CoV-2/inmunología , Vacunación/estadística & datos numéricos , Adulto , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Femenino , Francia/epidemiología , Hospitales Universitarios , Humanos , Memoria Inmunológica/inmunología , Incidencia , Masculino , Células B de Memoria/inmunología , Células T de Memoria/inmunología , Persona de Mediana Edad , Glicoproteína de la Espiga del Coronavirus/inmunología
2.
Proc Natl Acad Sci U S A ; 121(16): e2314426121, 2024 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-38574017

RESUMEN

Epstein-Barr Virus (EBV) infects more than 90% of the adult population worldwide. EBV infection is associated with Burkitt lymphoma (BL) though alone is not sufficient to induce carcinogenesis implying the involvement of co-factors. BL is endemic in African regions faced with mycotoxins exposure. Exposure to mycotoxins and oncogenic viruses has been shown to increase cancer risks partly through the deregulation of the immune response. A recent transcriptome profiling of B cells exposed to aflatoxin B1 (AFB1) revealed an upregulation of the Chemokine ligand 22 (CCL22) expression although the underlying mechanisms were not investigated. Here, we tested whether mycotoxins and EBV exposure may together contribute to endemic BL (eBL) carcinogenesis via immunomodulatory mechanisms involving CCL22. Our results revealed that B cells exposure to AFB1 and EBV synergistically stimulated CCL22 secretion via the activation of Nuclear Factor-kappa B pathway. By expressing EBV latent genes in B cells, we revealed that elevated levels of CCL22 result not only from the expression of the latent membrane protein LMP1 as previously reported but also from the expression of other viral latent genes. Importantly, CCL22 overexpression resulting from AFB1-exposure in vitro increased EBV infection through the activation of phosphoinositide-3-kinase pathway. Moreover, inhibiting CCL22 in vitro and in humanized mice in vivo limited EBV infection and decreased viral genes expression, supporting the notion that CCL22 overexpression plays an important role in B cell infection. These findings unravel new mechanisms that may underpin eBL development and identify novel pathways that can be targeted in drug development.


Asunto(s)
Linfoma de Burkitt , Infecciones por Virus de Epstein-Barr , Animales , Ratones , Herpesvirus Humano 4/genética , Infecciones por Virus de Epstein-Barr/complicaciones , Aflatoxina B1/toxicidad , Ligandos , Linfoma de Burkitt/metabolismo , Quimiocinas , Carcinogénesis
3.
J Biol Chem ; : 107849, 2024 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-39357828

RESUMEN

Several studies recently highlighted the role of lipoprotein receptors in viral entry. These receptors are evolutionarily ancient proteins, key for transport of lipids as well as other signaling molecules across the plasma membrane. Here, we discuss the different families of lipoprotein receptors and how they are hijacked by enveloped viruses to promote their entry into infected cells. While the usage of lipoprotein receptors was known for members of Flaviviridae family and for vesicular stomatitis virus, the last four years have seen the discovery that these receptors are used by many genetically unrelated viruses. We also emphasize how viral particles interact with these receptors and the possible targeting of these host factors as antiviral strategies.

4.
Traffic ; 23(1): 63-80, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34729868

RESUMEN

Lipid droplets (LDs) are involved in viral infections, but exactly how remains unclear. Here, we study the hepatitis C virus (HCV) whose core capsid protein binds to LDs but is also involved in the assembly of virions at the endoplasmic reticulum (ER) bilayer. We found that the amphipathic helix-containing domain of core, D2, senses triglycerides (TGs) rather than LDs per se. In the absence of LDs, D2 can bind to the ER membrane but only if TG molecules are present in the bilayer. Accordingly, the pharmacological inhibition of the diacylglycerol O-acyltransferase enzymes, mediating TG synthesis in the ER, inhibits D2 association with the bilayer. We found that TG molecules enable D2 to fold into alpha helices. Sequence analysis reveals that D2 resembles the apoE lipid-binding region. Our data support that TG in LDs promotes the folding of core, which subsequently relocalizes to contiguous ER regions. During this motion, core may carry TG molecules to these regions where HCV lipoviroparticles likely assemble. Consistent with this model, the inhibition of Arf1/COPI, which decreases LD surface accessibility to proteins and ER-LD material exchange, severely impedes the assembly of virions. Altogether, our data uncover a critical function of TG in the folding of core and HCV replication and reveals, more broadly, how TG accumulation in the ER may provoke the binding of soluble amphipathic helix-containing proteins to the ER bilayer.


Asunto(s)
Retículo Endoplásmico , Hepatitis C , Retículo Endoplásmico/metabolismo , Hepacivirus/fisiología , Hepatitis C/metabolismo , Humanos , Gotas Lipídicas/metabolismo , Triglicéridos/metabolismo , Proteínas del Núcleo Viral/metabolismo
5.
J Hepatol ; 78(5): 958-970, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36702177

RESUMEN

BACKGROUND & AIMS: Chronic coinfection with HBV and HDV leads to the most aggressive form of chronic viral hepatitis. Herein, we aimed to elucidate the molecular mechanisms underlying the widely reported observation that HDV interferes with HBV in most coinfected patients. METHODS: Patient liver tissues, primary human hepatocytes, HepaRG cells and human liver chimeric mice were used to analyze the effect of HDV on HBV using virological and RNA-sequencing analyses, as well as RNA synthesis, stability and association assays. RESULTS: Transcriptomic analyses in cell culture and mouse models of coinfection enabled us to define an HDV-induced signature, mainly composed of interferon (IFN)-stimulated genes (ISGs). We also provide evidence that ISGs are upregulated in chronically HDV/HBV-coinfected patients but not in cells that only express HDV antigen (HDAg). Inhibition of the hepatocyte IFN response partially rescued the levels of HBV parameters. We observed less HBV RNA synthesis upon HDV infection or HDV protein expression. Additionally, HDV infection or expression of HDAg alone specifically accelerated the decay of HBV RNA, and HDAg was associated with HBV RNAs. On the contrary, HDAg expression did not affect other viruses such as HCV or SARS-CoV-2. CONCLUSIONS: Our data indicate that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms. Specifically, we uncover a new viral interference mechanism in which proteins of a satellite virus affect the RNA production of its helper virus. Exploiting these findings could pave the way to the development of new therapeutic strategies against HBV. IMPACT AND IMPLICATIONS: Although the molecular mechanisms remained unexplored, it has long been known that despite its dependency, HDV decreases HBV viremia in patients. Herein, using in vitro and in vivo models, we showed that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms affecting HBV RNA metabolism, and we defined the HDV-induced modulation signature. The mechanisms we uncovered could pave the way for the development of new therapeutic strategies against HBV by mimicking and/or increasing the effect of HDAg on HBV RNA. Additionally, the HDV-induced modulation signature could potentially be correlated with responsiveness to IFN-α treatment, thereby helping to guide management of HBV/HDV-coinfected patients.


Asunto(s)
COVID-19 , Coinfección , Hepatitis B , Hepatitis D , Humanos , Ratones , Animales , Virus de la Hepatitis Delta/fisiología , Virus de la Hepatitis B/fisiología , Interferones , Antígenos de Hepatitis delta/metabolismo , Hepatitis D/complicaciones , Hepatitis B/complicaciones , Replicación Viral/fisiología , COVID-19/complicaciones , SARS-CoV-2/genética , ARN Viral/genética
6.
J Biol Chem ; 296: 100111, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33229438

RESUMEN

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a ß-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins: spike (S), envelope (E), membrane (M), and nucleoprotein (N) proteins. The involvement of each of these proteins and their interactions are critical for assembly and production of ß-coronavirus particles. Here, we sought to characterize the interplay of SARS-CoV-2 structural proteins during the viral assembly process. By combining biochemical and imaging assays in infected versus transfected cells, we show that E and M regulate intracellular trafficking of S as well as its intracellular processing. Indeed, the imaging data reveal that S is relocalized at endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) or Golgi compartments upon coexpression of E or M, as observed in SARS-CoV-2-infected cells, which prevents syncytia formation. We show that a C-terminal retrieval motif in the cytoplasmic tail of S is required for its M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlight that E and M induce a specific maturation of N-glycosylation of S, independently of the regulation of its localization, with a profile that is observed both in infected cells and in purified viral particles. Finally, we show that E, M, and N are required for optimal production of virus-like-particles. Altogether, these results highlight how E and M proteins may influence the properties of S proteins and promote the assembly of SARS-CoV-2 viral particles.


Asunto(s)
Proteínas de la Envoltura de Coronavirus/genética , Proteínas de la Nucleocápside/genética , SARS-CoV-2/crecimiento & desarrollo , Glicoproteína de la Espiga del Coronavirus/genética , Proteínas de la Matriz Viral/genética , Virión/crecimiento & desarrollo , Ensamble de Virus/fisiología , Animales , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Línea Celular Tumoral , Chlorocebus aethiops , Proteínas de la Envoltura de Coronavirus/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Retículo Endoplásmico/virología , Expresión Génica , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Aparato de Golgi/virología , Células HEK293 , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Hepatocitos/virología , Interacciones Huésped-Patógeno/genética , Humanos , Proteínas de la Nucleocápside/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero , Proteínas de la Matriz Viral/metabolismo , Virión/genética , Virión/metabolismo , Internalización del Virus , Liberación del Virus/fisiología
7.
Cancer Immunol Immunother ; 71(7): 1771-1775, 2022 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-34748076

RESUMEN

Here, we report a novel experimental setup to perform adoptive transfer of gene-edited B cells using humanized immune system mice by infusing autologous HIS mouse-derived human B cells "educated" in a murine context and thus rendered tolerant to the host. The present approach presents two advantages over the conventional humanized PBMC mouse models: (i) it circumvents the risk of xenogeneic graft-versus-host reaction and (ii) it mimics more closely human immune responses, thus favoring clinical translation. We show that the frequencies and numbers of transduced B cells in recipient's spleens one week post-transfer are within the range of the size of the pre-immune B cell population specific for a given protein antigen in the mouse. They are also compatible with the B cell numbers required to elicit a sizeable immune response upon immunization. Altogether, our findings pave the way for future studies aiming at assessing therapeutic interventions involving B cell reprogramming for instance by an antibody transgene in a "humanized" hematopoietic setting.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Leucocitos Mononucleares , Traslado Adoptivo , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones SCID
8.
PLoS Pathog ; 16(9): e1008850, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32956404

RESUMEN

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus that has become a serious threat to the public health. CCHFV has a single-stranded, tripartite RNA genome composed of L, M, and S segments. Cleavage of the M polyprotein precursor generates the two envelope glycoproteins (GPs) as well as three secreted nonstructural proteins GP38 and GP85 or GP160, representing GP38 only or GP38 linked to a mucin-like protein (MLD), and a double-membrane-spanning protein called NSm. Here, we examined the relevance of each M-segment non-structural proteins in virus assembly, egress and infectivity using a well-established CCHFV virus-like-particle system (tc-VLP). Deletion of MLD protein had no impact on infectivity although it reduced by 60% incorporation of GPs into particles. Additional deletion of GP38 abolished production of infectious tc-VLPs. The loss of infectivity was associated with impaired Gc maturation and exclusion from the Golgi, showing that Gn is not sufficient to target CCHFV GPs to the site of assembly. Consistent with this, efficient complementation was achieved in cells expressing MLD-GP38 in trans with increased levels of preGc to Gc conversion, co-targeting to the Golgi, resulting in particle incorporation and restored infectivity. Contrastingly, a MLD-GP38 variant retained in the ER allowed preGc cleavage but failed to rescue miss-localization or infectivity. NSm deletion, conversely, did not affect trafficking of Gc but interfered with Gc processing, particle formation and secretion. NSm expression affected N-glycosylation of different viral proteins most likely due to increased speed of trafficking through the secretory pathway. This highlights a potential role of NSm in overcoming Golgi retention and facilitating CCHFV egress. Thus, deletions of GP38 or NSm demonstrate their important role on CCHFV particle production and infectivity. GP85 is an essential viral factor for preGc cleavage, trafficking and Gc incorporation into particles, whereas NSm protein is involved in CCHFV assembly and virion secretion.


Asunto(s)
Virus de la Fiebre Hemorrágica de Crimea-Congo/fisiología , Proteínas Estructurales Virales , Ensamble de Virus , Línea Celular Tumoral , Eliminación de Gen , Humanos , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismo
9.
PLoS Pathog ; 16(11): e1008593, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-33180834

RESUMEN

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.


Asunto(s)
Carcinoma Hepatocelular/virología , Proteínas de Ciclo Celular/metabolismo , Virus de la Hepatitis B/fisiología , Hepatitis B/virología , Neoplasias Hepáticas/virología , Proteínas Represoras/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Proteínas del Núcleo Viral/metabolismo , Proteínas de Ciclo Celular/genética , Virus de la Hepatitis B/genética , Hepatocitos/virología , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Proteómica , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/genética , Factores de Empalme Serina-Arginina/genética , Proteínas del Núcleo Viral/genética , Replicación Viral
10.
J Biol Chem ; 295(45): 15196-15197, 2020 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-33158917

RESUMEN

The penetration of enveloped viruses into target cells requires the fusion of the lipid envelope of their virions with the host lipid membrane though a stepwise and highly sophisticated process. However, the intermediate steps in this process have seldom been visualized due to their rarity and rapidity. Here, using cryo-electron tomography, TIRF microscopy, and cell membrane-derived vesicles called blebs, Ward et al. visualize intermediates of the HIV-cell membrane fusion process and demonstrate how Serinc proteins prevent full fusion by interfering with this process.


Asunto(s)
Infecciones por VIH , Fusión de Membrana , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Electrones , Humanos , Virión
11.
J Biol Chem ; 295(7): 1843-1856, 2020 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-31929110

RESUMEN

Viruses depend on the host cell translation machinery for their replication, and one common strategy is the presence of internal ribosome entry sites (IRESs) in the viral RNAs, using different sets of host translation initiation factors. The hepatitis C virus (HCV) IRES binds eukaryotic translation initiation factor 3 (eIF3), but the exact functional role of the eIF3 complex and of its subunits remains to be precisely defined. Toward this goal, here we focused on eIF3 subunit e. We used an in vitro assay combining a ribosome-depleted rabbit reticulocyte lysate and ribosomes prepared from HeLa or Huh-7.5 cells transfected with either control or eIF3e siRNAs. eIF3e silencing reduced translation mediated by the 5'UTR of various cellular genes and HCV-like IRESs. However, this effect was not observed with the bona fide HCV IRES. Silencing of eIF3e reduced the intracellular levels of the c, d, and l subunits of eIF3 and their association with the eIF3 core subunit a. A pulldown analysis of eIF3 subunits associated with the HCV IRES disclosed similar effects and that the a subunit is critical for binding to the HCV IRES. Carrying out HCV infections of control and eIF3e-silenced Huh-7.5 cells, we found that in agreement with the in vitro findings, eIF3e silencing does not reduce HCV replication and viral protein expression. We conclude that unlike for host cellular mRNAs, the entire eIF3 is not required for HCV RNA translation, favoring viral expression under conditions of low eIF3e levels.


Asunto(s)
Factor 3 de Iniciación Eucariótica/genética , Hepacivirus/genética , Hepatitis C/genética , Sitios Internos de Entrada al Ribosoma/genética , Animales , Línea Celular , Hepacivirus/patogenicidad , Hepatitis C/patología , Hepatitis C/virología , Humanos , Unión Proteica/genética , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN Viral/química , ARN Viral/genética , Conejos , Ribosomas/química , Ribosomas/genética , Proteínas Virales/química , Proteínas Virales/genética
12.
PLoS Pathog ; 15(2): e1007589, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30818370

RESUMEN

Human T Lymphotropic virus (HTLV) infection can persist in individuals resulting, at least in part, from viral escape of the innate immunity, including inhibition of type I interferon response in infected T-cells. Plasmacytoid dendritic cells (pDCs) are known to bypass viral escape by their robust type I interferon production. Here, we demonstrated that pDCs produce type I interferons upon physical cell contact with HTLV-infected cells, yet pDC activation inversely correlates with the ability of the HTLV-producing cells to transmit infection. We show that pDCs sense surface associated-HTLV present with glycan-rich structure referred to as biofilm-like structure, which thus represents a newly described viral structure triggering the antiviral response by pDCs. Consistently, heparan sulfate proteoglycans and especially the cell surface pattern of terminal ß-galactoside glycosylation, modulate the transmission of the immunostimulatory RNA to pDCs. Altogether, our results uncover a function of virus-containing cell surface-associated glycosylated structures in the activation of innate immunity.


Asunto(s)
Células Dendríticas/fisiología , Infecciones por HTLV-I/metabolismo , Citocinas , Galactósidos/metabolismo , Glicosilación , Infecciones por HTLV-I/inmunología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Virus Linfotrópico T Tipo 2 Humano/inmunología , Virus Linfotrópico T Tipo 2 Humano/patogenicidad , Humanos , Inmunidad Innata/fisiología , Interferón Tipo I/inmunología , Interferón-alfa/inmunología , Interferón-alfa/metabolismo , Células Jurkat , Linfocitos T/inmunología , Linfocitos T/fisiología
13.
Int J Mol Sci ; 22(18)2021 Sep 16.
Artículo en Inglés | MEDLINE | ID: mdl-34576154

RESUMEN

Nowadays, cancers still represent a significant health burden, accounting for around 10 million deaths per year, due to ageing populations and inefficient treatments for some refractory cancers. Immunotherapy strategies that modulate the patient's immune system have emerged as good treatment options. Among them, the adoptive transfer of B cells selected ex vivo showed promising results, with a reduction in tumor growth in several cancer mouse models, often associated with antitumoral immune responses. Aside from the benefits of their intrinsic properties, including antigen presentation, antibody secretion, homing and long-term persistence, B cells can be modified prior to reinfusion to increase their therapeutic role. For instance, B cells have been modified mainly to boost their immuno-stimulatory activation potential by forcing the expression of costimulatory ligands using defined culture conditions or gene insertion. Moreover, tumor-specific antigen presentation by infused B cells has been increased by ex vivo antigen loading (peptides, RNA, DNA, virus) or by the sorting/ engineering of B cells with a B cell receptor specific to tumor antigens. Editing of the BCR also rewires B cell specificity toward tumor antigens, and may trigger, upon antigen recognition, the secretion of antitumor antibodies by differentiated plasma cells that can then be recognized by other immune components or cells involved in tumor clearance by antibody-dependent cell cytotoxicity or complement-dependent cytotoxicity for example. With the expansion of gene editing methodologies, new strategies to reprogram immune cells with whole synthetic circuits are being explored: modified B cells can sense disease-specific biomarkers and, in response, trigger the expression of therapeutic molecules, such as molecules that counteract the tumoral immunosuppressive microenvironment. Such strategies remain in their infancy for implementation in B cells, but are likely to expand in the coming years.


Asunto(s)
Linfocitos B/metabolismo , Edición Génica/métodos , Animales , Anticuerpos/metabolismo , Presentación de Antígeno/genética , Presentación de Antígeno/fisiología , Humanos , Inmunoterapia , Inmunoterapia Adoptiva/métodos
14.
J Virol ; 93(5)2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30541833

RESUMEN

Human hepatitis B virus (HBV) is a global health problem, affecting more than 250 million people worldwide. HBV-like viruses, named orthohepadnaviruses, also naturally infect nonhuman primates, rodents, and bats, but their pathogenicity and evolutionary history are unclear. Here, we determined the evolutionary history of the HBV receptors NTCP and GPC5 over millions of years of primate, rodent, and bat evolution. We use this as a proxy to understand the pathogenicity of orthohepadnaviruses in mammalian hosts and to determine the implications for species specificity. We found that NTCP, but not GPC5, has evolved under positive selection in primates (27 species), rodents (18 species), and bats (21 species) although at distinct residues. Notably, the positively selected codons map to the HBV-binding sites in primate NTCP, suggesting past genetic "arms races" with pathogenic orthohepadnaviruses. In rodents, the positively selected codons fall outside and within the presumed HBV-binding sites, which may contribute to the restricted circulation of rodent orthohepadnaviruses. In contrast, the presumed HBV-binding motifs in bat NTCP are conserved, and none of the positively selected codons map to this region. This suggests that orthohepadnaviruses may bind to different surfaces in bat NTCP. Alternatively, the patterns may reflect adaptive changes associated with metabolism rather than pathogens. Overall, our findings further point to NTCP as a naturally occurring genetic barrier for cross-species transmissions in primates, which may contribute to the narrow host range of HBV. In contrast, this constraint seems less important in bats, which may correspond to greater orthohepadnavirus circulation and diversity.IMPORTANCE Chronic infection with hepatitis B virus (HBV) is a major cause of liver disease and cancer in humans. Mammalian HBV-like viruses are also found in nonhuman primates, rodents, and bats. As for most viruses, HBV requires a successful interaction with a host receptor for replication. Cellular receptors are thus key determinants of host susceptibility as well as specificity. One hallmark of pathogenic virus-host relationships is the reciprocal evolution of host receptor and viral envelope proteins, as a result of their antagonistic interaction over time. The dynamics of these so-called "evolutionary arms races" can leave signatures of adaptive selection, which in turn reveal the evolutionary history of the virus-host interaction as well as viral pathogenicity and the genetic determinants of species specificity. Here, we show how HBV-like viruses have shaped the evolutionary history of their mammalian host receptor, as a result of their ancient pathogenicity, and decipher the genetic determinants of cross-species transmissions.


Asunto(s)
Virus de la Hepatitis B/patogenicidad , Hepatitis B/veterinaria , Especificidad del Huésped/genética , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Simportadores/genética , Proteínas del Envoltorio Viral/genética , Animales , Quirópteros/virología , Evolución Molecular , Variación Genética , Hepatitis B/patología , Hepatitis B/virología , Humanos , Primates/virología , Roedores/virología , Especificidad de la Especie , Internalización del Virus
15.
PLoS Pathog ; 14(3): e1006908, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29505618

RESUMEN

Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses.


Asunto(s)
Evolución Molecular , Hepacivirus/fisiología , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Hepatitis C/metabolismo , Hepatitis C/patología , Hepatitis C/virología , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Células Tumorales Cultivadas , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Replicación Viral
16.
Hepatology ; 69(5): 2214-2231, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30549291

RESUMEN

The liver is an organ with strong regenerative capacity, yet primary hepatocytes have a low amplification potential in vitro, a major limitation for the cell-based therapy of liver disorders and for ex vivo biological screens. Induced pluripotent stem cells (iPSCs) may help to circumvent this obstacle but often harbor genetic and epigenetic abnormalities, limiting their potential. Here, we describe the pharmacological induction of proliferative human hepatic progenitor cells (HPCs) through a cocktail of growth factors and small molecules mimicking the signaling events involved in liver regeneration. Human HPCs from healthy donors and pediatric patients proliferated vigorously while maintaining their genomic stability and could be redifferentiated in vitro into metabolically competent cells that supported the replication of hepatitis B and delta viruses. Redifferentiation efficiency was boosted by three-dimensional culture. Finally, transcriptome analysis showed that HPCs were more closely related to mature hepatocytes than iPSC-derived hepatocyte-like cells were. Conclusion: HPC induction holds promise for a variety of applications such as ex vivo disease modeling, personalized drug testing or metabolic studies, and development of a bioartificial liver.


Asunto(s)
Técnicas de Cultivo de Célula , Medios de Cultivo/química , Hepatocitos/fisiología , Hígado/citología , Células Madre , Animales , Estudios de Casos y Controles , Masculino , Ratones Endogámicos NOD , Cultivo Primario de Células
17.
FASEB J ; 33(2): 2472-2483, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30307769

RESUMEN

Hepatitis B virus (HBV) infection and bile acid (BA) metabolism are interdependent: infection modifies the expression of the BA nuclear receptor farnesoid X receptor (FXR)-α, and modulation of FXRα activity by ligands alters HBV replication. Mechanisms of HBV control by FXRα remain to be unveiled. FXRα silencing in HBV-infected HepaRG cells decreased the viral covalently closed circular (ccc)DNA pool size and transcriptional activity. Treatment with the FXRα agonist GW4064 inhibited FXRα proviral effect on cccDNA similarly for wild-type and hepatitis B viral X protein (HBx)-deficient virus, whereas agonist-induced inhibition of pregenomic and precore RNA transcription and viral DNA secretion was HBx dependent. These data indicated that FXRα acts as a proviral factor by 2 different mechanisms, which are abolished by FXRα stimulation. Finally, infection of C3H/HeN mice by a recombinant adeno-associated virus-2/8-HBV vector induced a sustained HBV replication in young mice in contrast with the transient decline in adult mice. Four-week GW4064 treatment of infected C3H/HeN mice decreased secretion of HBV DNA and HB surface antigen in adult mice only. These results suggest that the physiologic balance of FXRα expression and activation by bile acid is a key host metabolic pathway in the regulation of HBV infection and that FXRα can be envisioned as a target for HBV treatment.-Mouzannar, K., Fusil, F., Lacombe, B., Ollivier, A., Ménard, C., Lotteau, V., Cosset, F.-L., Ramière, C., André, P. Farnesoid X receptor α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo.


Asunto(s)
Regulación de la Expresión Génica , Virus de la Hepatitis B/patogenicidad , Hepatitis B/virología , Provirus/patogenicidad , Receptores Citoplasmáticos y Nucleares/metabolismo , Replicación Viral , Animales , ADN Viral/genética , Femenino , Células Hep G2 , Hepatitis B/metabolismo , Hepatitis B/patología , Virus de la Hepatitis B/genética , Humanos , Técnicas In Vitro , Ligandos , Ratones , Ratones Endogámicos C3H , Provirus/genética
18.
J Hepatol ; 70(4): 626-638, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30553840

RESUMEN

BACKGROUND & AIMS: In the sera of infected patients, hepatitis C virus (HCV) particles display heterogeneous forms with low-buoyant densities (<1.08), underscoring their lipidation via association with apoB-containing lipoproteins, which was proposed to occur during assembly or secretion from infected hepatocytes. However, the mechanisms inducing this association remain poorly-defined and most cell culture grown HCV (HCVcc) particles exhibit higher density (>1.08) and poor/no association with apoB. We aimed to elucidate the mechanisms of lipidation and to produce HCVcc particles resembling those in infected sera. METHODS: We produced HCVcc particles of Jc1 or H77 strains from Huh-7.5 hepatoma cells cultured in standard conditions (10%-fetal calf serum) vs. in serum-free or human serum conditions before comparing their density profiles to patient-derived virus. We also characterized wild-type and Jc1/H77 hypervariable region 1 (HVR1)-swapped mutant HCVcc particles produced in serum-free media and incubated with different serum types or with purified lipoproteins. RESULTS: Compared to serum-free or fetal calf serum conditions, production with human serum redistributed most HCVcc infectious particles to low density (<1.08) or very-low density (<1.04) ranges. In addition, short-time incubation with human serum was sufficient to shift HCVcc physical particles to low-density fractions, in time- and dose-dependent manners, which increased their specific infectivity, promoted apoB-association and induced neutralization-resistance. Moreover, compared to Jc1, we detected higher levels of H77 HCVcc infectious particles in very-low-density fractions, which could unambiguously be attributed to strain-specific features of the HVR1 sequence. Finally, all 3 lipoprotein classes, i.e., very-low-density, low-density and high-density lipoproteins, could synergistically induce low-density shift of HCV particles; yet, this required additional non-lipid serum factor(s) that include albumin. CONCLUSIONS: The association of HCV particles with lipids may occur in the extracellular milieu. The lipidation level depends on serum composition as well as on HVR1-specific properties. These simple culture conditions allow production of infectious HCV particles resembling those of chronically-infected patients. LAY SUMMARY: Hepatitis C virus (HCV) particles may associate with apoB and acquire neutral lipids after exiting cells, giving them low-buoyant density. The hypervariable region 1 (HVR1) is a majorviral determinant of E2 that controls this process. Besides lipoproteins, specific serum factors including albumin promote extracellular maturation of HCV virions. HCV particle production in vitro, with media of defined serum conditions, enables production of infectious particles resembling those of chronically infected patients.


Asunto(s)
Apolipoproteína B-100/metabolismo , Líquido Extracelular/metabolismo , Hepacivirus/metabolismo , Hepatitis C/metabolismo , Albúmina Sérica Humana/metabolismo , Virión/metabolismo , Apolipoproteínas E/metabolismo , Línea Celular Tumoral , HDL-Colesterol/metabolismo , LDL-Colesterol/metabolismo , VLDL-Colesterol/metabolismo , Medios de Cultivo Condicionados , Medio de Cultivo Libre de Suero , Hepatitis C/virología , Hepatocitos/metabolismo , Hepatocitos/virología , Humanos , Proteínas del Envoltorio Viral/química , Proteínas Virales/metabolismo , Ensamble de Virus
19.
PLoS Pathog ; 13(12): e1006774, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-29253880

RESUMEN

Viroporins are small transmembrane proteins with ion channel activities modulating properties of intracellular membranes that have diverse proviral functions. Hepatitis C virus (HCV) encodes a viroporin, p7, acting during assembly, envelopment and secretion of viral particles (VP). HCV p7 is released from the viral polyprotein through cleavage at E2-p7 and p7-NS2 junctions by signal peptidase, but also exists as an E2p7 precursor, of poorly defined properties. Here, we found that ectopic p7 expression in HCVcc-infected cells reduced secretion of particle-associated E2 glycoproteins. Using biochemical assays, we show that p7 dose-dependently slows down the ER-to-Golgi traffic, leading to intracellular retention of E2, which suggested that timely E2p7 cleavage and p7 liberation are critical events to control E2 levels. By studying HCV mutants with accelerated E2p7 processing, we demonstrate that E2p7 cleavage controls E2 intracellular expression and secretion levels of nucleocapsid-free subviral particles and infectious virions. In addition, our imaging data reveal that, following p7 liberation, the amino-terminus of p7 is exposed towards the cytosol and coordinates the encounter between NS5A and NS2-based assembly sites loaded with E1E2 glycoproteins, which subsequently leads to nucleocapsid envelopment. We identify punctual mutants at p7 membrane interface that, by abrogating NS2/NS5A interaction, are defective for transmission of infectivity owing to decreased secretion of core and RNA and to increased secretion of non/partially-enveloped particles. Altogether, our results indicate that the retarded E2p7 precursor cleavage is essential to regulate the intracellular and secreted levels of E2 through p7-mediated modulation of the cell secretory pathway and to unmask critical novel assembly functions located at p7 amino-terminus.


Asunto(s)
Hepacivirus/fisiología , Hepacivirus/patogenicidad , Proteínas del Envoltorio Viral/fisiología , Proteínas Virales/fisiología , Secuencia de Aminoácidos , Línea Celular , Células HEK293 , Hepacivirus/genética , Hepatitis C/etiología , Hepatitis C/virología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Humanos , Modelos Biológicos , Mutación , Procesamiento Proteico-Postraduccional , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas no Estructurales Virales/fisiología , Proteínas Virales/química , Proteínas Virales/genética , Virulencia/genética , Virulencia/fisiología , Ensamble de Virus/genética , Ensamble de Virus/fisiología
20.
Gastroenterology ; 152(4): 895-907.e14, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-27932311

RESUMEN

BACKGROUND & AIMS: Daclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs, and inhibition of viral particle assembly. METHODS: Cell-culture-derived HCV particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity and production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy. RESULTS: Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B, and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes. CONCLUSIONS: In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins because viral particles and replication complex vesicles cannot form or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection.


Asunto(s)
Antivirales/farmacología , Genoma Viral , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Imidazoles/farmacología , Transporte de ARN/efectos de los fármacos , ARN Viral/metabolismo , Carbamatos , Línea Celular Tumoral , Hepacivirus/fisiología , Humanos , Transporte de Proteínas/efectos de los fármacos , Pirrolidinas , Valina/análogos & derivados , Proteínas del Núcleo Viral/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas no Estructurales Virales/metabolismo , Ensamble de Virus/efectos de los fármacos
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