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In order to survive and replicate, Salmonella has evolved mechanisms to gain access to intestinal epithelial cells of the crypt. However, the impact of Salmonella Typhimurium on stem cells and progenitors, which are responsible for the ability of the intestinal epithelium to renew and protect itself, remains unclear. Given that intestinal organoids growth is sustained by stem cells and progenitors activity, we have used this model to document the effects of Salmonella Typhimurium infection on epithelial proliferation and differentiation, and compared it to an in vivo model of Salmonella infection in mice. Among gut segments, the caecum was preferentially targeted by Salmonella. Analysis of infected crypts and organoids demonstrated increased length and size, respectively. mRNA transcription profiles of infected crypts and organoids pointed to upregulated EGFR-dependent signals, associated with a decrease in secretory cell lineage differentiation. To conclude, we show that organoids are suited to mimic the impact of Salmonella on stem cells and progenitors cells, carrying a great potential to drastically reduce the use of animals for scientific studies on that topic. In both models, the EGFR pathway, crucial to stem cells and progenitors proliferation and differentiation, is dysregulated by Salmonella, suggesting that repeated infections might have consequences on crypt integrity and further oncogenesis.
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Diferenciación Celular , Receptores ErbB , Organoides , Infecciones por Salmonella , Salmonella typhimurium , Células Madre , Animales , Organoides/microbiología , Células Madre/metabolismo , Ratones , Salmonella typhimurium/patogenicidad , Salmonella typhimurium/fisiología , Infecciones por Salmonella/microbiología , Infecciones por Salmonella/patología , Receptores ErbB/metabolismo , Receptores ErbB/genética , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Proliferación Celular , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
The Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging pathogen of the Orthonairovirus genus that can cause severe and often lethal hemorrhagic diseases in humans. CCHFV has a broad tropism and can infect a variety of species and tissues. Here, by using gene silencing, blocking antibodies or soluble receptor fragments, we identify the low-density lipoprotein receptor (LDL-R) as a CCHFV entry factor. The LDL-R facilitates binding of CCHFV particles but does not allow entry of Hazara virus (HAZV), another member of the genus. In addition, we show that apolipoprotein E (apoE), an exchangeable protein that mediates LDL/LDL-R interaction, is incorporated on CCHFV particles, though not on HAZV particles, and enhances their specific infectivity by promoting an LDL-R dependent entry. Finally, we show that molecules that decrease LDL-R from the surface of target cells could inhibit CCHFV infection. Our study highlights that CCHFV takes advantage of a lipoprotein receptor and recruits its natural ligand to promote entry into cells.
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Apolipoproteínas E , Virus de la Fiebre Hemorrágica de Crimea-Congo , Receptores de LDL , Internalización del Virus , Humanos , Receptores de LDL/metabolismo , Apolipoproteínas E/metabolismo , Apolipoproteínas E/genética , Virus de la Fiebre Hemorrágica de Crimea-Congo/metabolismo , Virus de la Fiebre Hemorrágica de Crimea-Congo/fisiología , Animales , Células HEK293 , Chlorocebus aethiops , Fiebre Hemorrágica de Crimea/virología , Fiebre Hemorrágica de Crimea/metabolismo , Virión/metabolismo , Células VeroRESUMEN
Tumor spheroids are promising three-dimensional (3D) in vitro tumor models for the evaluation of drug delivery methods. The design of noninvasive and targeted drug methods is required to improve the intratumoral bioavailability of chemotherapeutic drugs and reduce their adverse off-target effects. Among such methods, microbubble-assisted ultrasound (MB-assisted US) is an innovative modality for noninvasive targeted drug delivery. The aim of the present study is to evaluate the efficacy of this US modality for the delivery of bleomycin, doxorubicin, and irinotecan in colorectal cancer (CRC) spheroids. MB-assisted US permeabilized the CRC spheroids to propidium iodide, which was used as a drug model without affecting their growth and viability. Histological analysis and electron microscopy revealed that MB-assisted US affected only the peripheral layer of the CRC spheroids. The acoustically mediated bleomycin delivery induced a significant decrease in CRC spheroid growth in comparison to spheroids treated with bleomycin alone. However, this US modality did not improve the therapeutic efficacy of doxorubicin and irinotecan on CRC spheroids. In conclusion, this study demonstrates that tumor spheroids are a relevant approach to evaluate the efficacy of MB-assisted US for the delivery of chemotherapeutics.
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Antineoplásicos , Neoplasias , Humanos , Irinotecán , Microburbujas , Doxorrubicina/farmacología , Bleomicina , Esferoides Celulares , Línea Celular TumoralRESUMEN
The coronavirus' (CoV) membrane (M) protein is the driving force during assembly, but this process remains poorly characterized. Previously, we described two motifs in the C-tail of the Middle East respiratory syndrome CoV (MERS-CoV) M protein involved in its endoplasmic reticulum (ER) exit (211DxE213) and trans-Golgi network (TGN) retention (199KxGxYR204). Here, their function in virus assembly was investigated by two different virus-like particle (VLP) assays and by mutating both motifs in an infectious MERS-CoV cDNA clone. It was shown that the 199KxGxYR204 motif was essential for VLP and infectious virus assembly. Moreover, the mislocalization of the M protein induced by mutation of this motif prevented M-E interaction. Hampering the ER export of M by mutating its 211DxE213 motif still allowed the formation of nucleocapsid-empty VLPs, but prevented the formation of fully assembled VLPs and infectious particles. Taken together, these data show that the MERS-CoV assembly process highly depends on the correct intracellular trafficking of its M protein, and hence that not only specific protein-protein interacting motifs but also correct subcellular localization of the M protein in infected cells is essential for virus formation and should be taken into consideration when studying the assembly process.
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Proteínas de la Membrana , Coronavirus del Síndrome Respiratorio de Oriente Medio , Proteínas de la Membrana/metabolismo , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Ensamble de Virus/genéticaRESUMEN
The human T-lymphotropic virus type 1 (HTLV-1) is an oncogenic retrovirus whose transmission relies primarily on cell-to-cell contacts as cell-free viruses are poorly infectious. Among the intercellular transmission routes described, HTLV-1 biofilms are adhesive structures polarized at the cell surface that confine virions in a protective environment, which is believed to promote their simultaneous delivery during infection. Here, we show that several tetraspanins are enriched in HTLV-1 biofilms and incorporated into the viral envelope. However, we report that only the tetraspanin CD82 interacts with HTLV-1 Gag proteins which initiates their polarization into viral biofilms. Also, we demonstrate that CD82 maintains HTLV-1 biofilm polarization and favors viral transmission, as its silencing induces a complete reorganization of viral clusters at the cell surface and reduces the ability of infected T-cells to transmit the virus. Our results highlight the crucial role of CD82 and its glycosylation state in the architectural organization of HTLV-1 biofilms and their subsequent transfer through intercellular contacts.IMPORTANCEIn the early stages of infection, human T-lymphotropic virus type 1 (HTLV-1) dissemination within its host is believed to rely mostly on cell-to-cell contacts. Past studies unveiled a novel mechanism of HTLV-1 intercellular transmission based on the remodeling of the host-cell extracellular matrix and the generation of cell-surface viral assemblies whose structure, composition, and function resemble bacterial biofilms. These polarized aggregates of infectious virions, identified as viral biofilms, allow the bulk delivery of viruses to target cells and may help to protect virions from immune attacks. However, viral biofilms' molecular and functional description is still in its infancy, although it is crucial to fully decipher retrovirus pathogenesis. Here, we explore the function of cellular tetraspanins (CD9, CD81, CD82) that we detect inside HTLV-1 particles within biofilms. Our results demonstrate specific roles for CD82 in the cell-surface distribution and intercellular transmission of HTLV-1 biofilms, which we document as two essential parameters for efficient viral transmission. At last, our findings indicate that N-glycosylation of cell-surface molecules, including CD82, is required for the polarization of HTLV-1 biofilms and for the efficient transmission of HTLV-1 between T-lymphocytes.
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IFITMs are a family of highly related interferon-induced transmembrane proteins that interfere with the processes of fusion between viral and cellular membranes and are thus endowed with broad antiviral properties. A number of studies have shown how the antiviral potency of IFITMs is highly dependent on their steady-state levels, their intracellular distribution and a complex pattern of post-translational modifications, parameters that are overall tributary of a number of cellular partners. In an effort to identify additional protein partners involved in the biology of IFITMs, we devised a proteomics-based approach based on the piggyback incorporation of IFITM3 partners into extracellular vesicles. MS analysis of the proteome of vesicles bearing or not bearing IFITM3 identified the NDFIP2 protein adaptor protein as an important regulator of IFITM3 levels. NDFIP2 is a membrane-anchored adaptor protein of the E3 ubiquitin ligases of the NEDD4 family that have already been found to be involved in IFITM3 regulation. We show here that NDFIP2 acts as a recruitment factor for both IFITM3 and NEDD4 and mediates their distribution in lysosomal vesicles. The genetic inactivation and overexpression of NDFIP2 drive, respectively, lower and higher levels of IFITM3 accumulation in the cell, overall suggesting that NDFIP2 locally competes with IFITM3 for NEDD4 binding. Given that NDFIP2 is itself tightly regulated and highly responsive to external cues, our study sheds light on a novel and likely dynamic layer of regulation of IFITM3.
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Proteómica , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Procesamiento Proteico-Postraduccional , Lisosomas/metabolismo , Antivirales/metabolismoRESUMEN
Chicken infection with Salmonella Typhimurium is an important source of foodborne human diseases. Salmonella colonizes the avian intestinal tract and more particularly the caecum, without causing symptoms. This thus poses a challenge for the prevention of foodborne transmission. Until now, studies on the interaction of Salmonella with the avian gut intestine have been limited by the absence of in vitro intestinal culture models. Here, we established intestinal crypt-derived chicken organoids to better decipher the impact of Salmonella intracellular replication on avian intestinal epithelium. Using a 3D organoid model, we observed a significantly higher replication rate of the intracellular bacteria in caecal organoids than in ileal organoids. Our model thus recreates intracellular environment, allowing Salmonella replication of avian epithelium according to the intestinal segment. Moreover, an inhibition of the cellular proliferation was observed in infected ileal and caecal organoids compared to uninfected organoids. This appears with a higher effect in ileal organoids, as well as a higher cytokine and signaling molecule response in infected ileal organoids at 3 h post-infection (hpi) than in caecal organoids that could explain the lower replication rate of Salmonella observed later at 24 hpi. To conclude, this study demonstrates that the 3D organoid is a model allowing to decipher the intracellular impact of Salmonella on the intestinal epithelium cell response and illustrates the importance of the gut segment used to purify stem cells and derive organoids to specifically study epithelial cell -Salmonella interaction.
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Pollos , Salmonella typhimurium , Humanos , Animales , Salmonella typhimurium/fisiología , Intestinos , Mucosa Intestinal/microbiología , Ciego , Organoides/microbiologíaRESUMEN
Recent evidence showed that in response to elevated sodium dietary intakes, many body tissues retain Na+ ions for long periods of time and can reach concentrations up to 200 mM. This could modulate the immune system and be responsible for several diseases. However, studies brought contrasted results and the effects of external sodium on human dendritic cell (DC) responses to danger signals remain largely unknown. Considering their central role in triggering T cell response, we tested how NaCl-enriched medium influences human DCs properties. We found that DCs submitted to high extracellular Na+ concentrations up to 200 mM remain viable and maintain the expression of specific DC markers, however, their maturation, chemotaxis toward CCL19, production of pro-inflammatory cytokines and ROS in response to LPS were also partially inhibited. In line with these results, the T-cell allostimulatory capacity of DCs was also inhibited. Finally, our data indicate that high NaCl concentrations triggered the phosphorylation of SGK1 and ERK1/2 kinases. These results raised the possibility that the previously reported pro-inflammatory effects of high NaCl concentrations on T cells might be counterbalanced by a downregulation of DC activation.
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Lipopolisacáridos , Cloruro de Sodio , Humanos , Cloruro de Sodio/farmacología , Cloruro de Sodio/metabolismo , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismo , Diferenciación Celular , Cloruros/metabolismo , Cloruros/farmacología , Células Dendríticas , Citocinas/metabolismo , Sodio/metabolismo , Sodio/farmacología , Células CultivadasRESUMEN
INTRODUCTION: Radial nerve palsy is a classical complication of a humeral shaft fracture. In clinical practice, motor palsy of the radial nerve is sometimes observed without an abnormality felt in the sensory territory. HYPOTHESIS: We hypothesised that this dissociation between sensory and motor involvement is related to anatomical variations of the sensory innervation of the dorsal surface of the first digit space, thus, we decided to study the nature and frequency of these variations. MATERIAL AND METHOD: A cadaveric study was conducted on 24 upper limbs to analyse the truncal origin of the sensory branches innervating the dorsal surface of the first digit space. RESULTS: The sensory branch of the radial nerve (SBRN) participated in the innervation of the dorsal surface of the first digit space in 22 limbs, an anatomical variation was present in 2 cases with a mixed innervation by the SBRN and the lateral cutaneous nerve of forearm (LCNF) in 1 case and singular innervation by LCNF, with no SBRN involvement, in 1 case. Communications between SBRN and LCNF were found in 7 cases. DISCUSSION: Pure motor radial damage, without a sensory deficit of the dorsal surface of the first digit space, does not preclude a complete traumatic injury of the radial nerve. The sensory innervation of this region can be relayed by a branch of the LCNF. LEVEL OF EVIDENCE: IV; cadaveric study.
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Antebrazo , Neuropatía Radial , Humanos , Antebrazo/inervación , Nervio Radial/anatomía & histología , Nervio Radial/lesiones , Pulgar , CadáverRESUMEN
Although hepatitis E virus (HEV) is the major leading cause of enterically transmitted viral hepatitis worldwide, many gaps remain in the understanding of the HEV lifecycle. Notably, viral factories induced by HEV have not been documented yet, and it is currently unknown whether HEV infection leads to cellular membrane modeling as many positive-strand RNA viruses. HEV genome encodes the ORF1 replicase, the ORF2 capsid protein and the ORF3 protein involved in virion egress. Previously, we demonstrated that HEV produces different ORF2 isoforms including the virion-associated ORF2i form. Here, we generated monoclonal antibodies that specifically recognize the ORF2i form and antibodies that recognize the different ORF2 isoforms. One antibody, named P1H1 and targeting the ORF2i N-terminus, recognized delipidated HEV particles from cell culture and patient sera. Importantly, AlphaFold2 modeling demonstrated that the P1H1 epitope is exposed on HEV particles. Next, antibodies were used to probe viral factories in HEV-producing/infected cells. By confocal microscopy, we identified subcellular nugget-like structures enriched in ORF1, ORF2 and ORF3 proteins and viral RNA. Electron microscopy analyses revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. We showed that these structures are dependent on ORF2i capsid protein assembly and ORF3 expression. An extensive colocalization study of viral proteins with subcellular markers, and silencing experiments demonstrated that these structures are derived from the endocytic recycling compartment (ERC) for which Rab11 is a central player. Hence, HEV hijacks the ERC and forms a membrane network of vesicular and tubular structures that might be the hallmark of HEV infection.
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Virus de la Hepatitis E , Humanos , Virus de la Hepatitis E/genética , Compartimentos de Replicación Viral , Proteínas de la Cápside , Transporte Biológico , Anticuerpos MonoclonalesRESUMEN
Zika virus (ZIKV) is a mosquito-borne flavivirus that emerged in the Pacific islands in 2007 and spread to the Americas in 2015. The infection remains asymptomatic in most cases but can be associated with severe neurological disorders. Despite massive efforts, no specific drug or vaccine against ZIKV infection is available to date. Claudins are tight-junction proteins that favor the entry of several flaviviruses, including ZIKV. In this study, we identified two peptides derived from the N-terminal sequences of claudin-7 and claudin-1, named CL7.1 and CL1.1, respectively, that inhibited ZIKV infection in a panel of human cell lines. Using cell-to-cell fusion assays, we demonstrated that these peptides blocked the ZIKV E-mediated membrane fusion. A comparison of the antiviral efficacy of CL1.1 and CL7.1 pointed to the importance of the peptide amphipathicity. Electron microscopic analysis revealed that CL1.1 altered the ultrastructure of the viral particles likely by binding the virus lipid envelope. However, amphipathicity could not fully explain the antiviral activity of CL1.1. In silico docking simulations suggested that CL1.1 may also interact with the E protein, near its stem region. Overall, our data suggested that claudin-derived peptides inhibition may be linked to simultaneous interaction with the E protein and the viral lipid envelope. Finally, we found that CL1.1 also blocked infection by yellow fever and Japanese encephalitis viruses but not by HIV-1 or SARS-CoV-2. Our results provide a basis for the future development of therapeutics against a wide range of endemic and emerging flaviviruses. IMPORTANCE Zika virus (ZIKV) is a flavivirus transmitted by mosquito bites that have spread to the Pacific Islands and the Americas over the past decade. The infection remains asymptomatic in most cases but can cause severe neurological disorders. ZIKV is a major public health threat in areas of endemicity, and there is currently no specific antiviral drug or vaccine available. We identified two antiviral peptides deriving from the N-terminal sequences of claudin-7 and claudin-1 with the latter being the most effective. These peptides block the envelope-mediated membrane fusion. Our data suggested that the inhibition was likely achieved by simultaneously interacting with the viral lipid envelope and the E protein. The peptides also inhibited other flaviviruses. These results could provide the basis for the development of therapies that might target a wide array of flaviviruses from current epidemics and possibly future emergences.
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Claudinas , Fusión de Membrana , Infección por el Virus Zika , Virus Zika , Humanos , Antivirales/farmacología , Claudina-1 , Lípidos , Péptidos/farmacología , Infección por el Virus Zika/tratamiento farmacológicoRESUMEN
BACKGROUND: Zika virus (ZIKV), a member of the Flaviviridae family, has caused massive outbreaks of infection in tropical areas over the last decade and has now begun spreading to temperate countries. Little is currently known about the specific host factors involved in the intracellular life cycle of ZIKV. Flaviviridae viruses interact closely with host-cell lipid metabolism and associated secretory pathways. Another Flaviviridae, hepatitis C virus, is highly dependent on apolipoprotein E (ApoE) for the completion of its infectious cycle. We therefore investigated whether ZIKV also interacted with this protein. METHODS: ZIKV infections were performed on both liver and microglia derived cell lines in order to proceed to colocalization analysis and immunoprecipitation assays of ApoE and Zika envelope glycoprotein (Zika E). Transmission electron microscopy combined to immunogold labeling was also performed on the infected cells and related supernatant to study the association of ApoE and Zika E protein in the virus-induced membrane rearrangements and secreted particles, respectively. Finally, the potential of neutralization of anti-ApoE antibodies on ZIKV particles was studied. RESULT: We demonstrated an interaction between ApoE and the Zika E protein. This specific interaction was observed in virus-induced host-cell membrane rearrangements, but also on newly formed intracellular particles. The partial neutralizing effect of anti-ApoE antibody and the immunogold labeling of the two proteins on secreted virions indicates that this interaction is conserved during ZIKV intracellular trafficking and release. CONCLUSIONS: These data suggest that another member of the Flaviviridae also interacts with ApoE, indicating that this could be a common mechanism for the viruses from this family.
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Flaviviridae , Infección por el Virus Zika , Virus Zika , Anticuerpos Antivirales , Apolipoproteínas E , Línea Celular , Humanos , Proteínas del Envoltorio Viral , Virión/metabolismoRESUMEN
Positive single-strand RNA (+ RNA) viruses can remodel host cell membranes to induce a replication organelle (RO) isolating the replication of their genome from innate immunity mechanisms. Some of these viruses, including severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), induce double-membrane vesicles (DMVs) for this purpose. Viral non-structural proteins are essential for DMV biogenesis, but they cannot form without an original membrane from a host cell organelle and a significant supply of lipids. The endoplasmic reticulum (ER) and the initial mechanisms of autophagic processes have been shown to be essential for the biogenesis of SARS-CoV-2 DMVs. However, by analogy with other DMV-inducing viruses, it seems likely that the Golgi apparatus, mitochondria and lipid droplets are also involved. As for hepatitis C virus (HCV), pores crossing both membranes of SARS-CoV-2-induced DMVs have been identified. These pores presumably allow the supply of metabolites essential for viral replication within the DMV, together with the export of the newly synthesized viral RNA to form the genome of future virions. It remains unknown whether, as for HCV, DMVs with open pores can coexist with the fully sealed DMVs required for the storage of large amounts of viral RNA. Interestingly, recent studies have revealed many similarities in the mechanisms of DMV biogenesis and morphology between these two phylogenetically distant viruses. An understanding of the mechanisms of DMV formation and their role in the infectious cycle of SARS-CoV-2 may be essential for the development of new antiviral approaches against this pathogen or other coronaviruses that may emerge in the future.
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COVID-19 , Hepatitis C , Retículo Endoplásmico/metabolismo , Hepacivirus/genética , Humanos , ARN Viral/genética , ARN Viral/metabolismo , SARS-CoV-2 , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
Infections of the central nervous system are among the most serious infections1,2, but the mechanisms by which pathogens access the brain remain poorly understood. The model microorganism Listeria monocytogenes (Lm) is a major foodborne pathogen that causes neurolisteriosis, one of the deadliest infections of the central nervous system3,4. Although immunosuppression is a well-established host risk factor for neurolisteriosis3,5, little is known about the bacterial factors that underlie the neuroinvasion of Lm. Here we develop a clinically relevant experimental model of neurolisteriosis, using hypervirulent neuroinvasive strains6 inoculated in a humanized mouse model of infection7, and we show that the bacterial surface protein InlB protects infected monocytes from Fas-mediated cell death by CD8+ T cells in a manner that depends on c-Met, PI3 kinase and FLIP. This blockade of specific anti-Lm cellular immune killing lengthens the lifespan of infected monocytes, and thereby favours the transfer of Lm from infected monocytes to the brain. The intracellular niche that is created by InlB-mediated cell-autonomous immune resistance also promotes Lm faecal shedding, which accounts for the selection of InlB as a core virulence gene of Lm. We have uncovered a specific mechanism by which a bacterial pathogen confers an increased lifespan to the cells it infects by rendering them resistant to cell-mediated immunity. This promotes the persistence of Lm within the host, its dissemination to the central nervous system and its transmission.
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Enfermedades del Sistema Nervioso Central , Listeria monocytogenes , Listeriosis , Animales , Proteínas Bacterianas/metabolismo , Linfocitos T CD8-positivos/metabolismo , Enfermedades del Sistema Nervioso Central/microbiología , Modelos Animales de Enfermedad , Listeria monocytogenes/patogenicidad , Listeriosis/microbiología , Ratones , Monocitos , VirulenciaRESUMEN
Listeria monocytogenes (Lm) is a foodborne pathogen and the etiological agent of listeriosis. This facultative intracellular Gram-positive bacterium has the ability to colonize the intestinal lumen, cross the intestinal, blood-brain and placental barriers, leading to bacteremia, neurolisteriosis and maternal-fetal listeriosis. Lm is a model microorganism for the study of the interplay between a pathogenic microbe, host tissues and microbiota in vivo. Here we review how animal models permissive to Lm-host interactions allow deciphering some of the key steps of the infectious process, from the intestinal lumen to the crossing of host barriers and dissemination within the host. We also highlight recent investigations using tagged Lm and clinically relevant strains that have shed light on within-host dynamics and the purifying selection of Lm virulence factors. Studying Lm infection in vivo is a way forward to explore host biology and unveil the mechanisms that have selected its capacity to closely associate with its vertebrate hosts.
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Listeria monocytogenes , Listeriosis , Animales , Proteínas Bacterianas , Femenino , Interacciones Huésped-Patógeno , Listeria monocytogenes/genética , Placenta , EmbarazoRESUMEN
In HIV, the polyprotein precursor Gag orchestrates the formation of the viral capsid. In the current view of this viral assembly, Gag forms low-order oligomers that bind to the viral genomic RNA triggering the formation of high-ordered ribonucleoprotein complexes. However, this assembly model was established using biochemical or imaging methods that do not describe the cellular location hosting Gag-gRNA complex nor distinguish gRNA packaging in single particles. Here, we studied the intracellular localization of these complexes by electron microscopy and monitored the distances between the two partners by morphometric analysis of gold beads specifically labeling Gag and gRNA. We found that formation of these viral clusters occurred shortly after the nuclear export of the gRNA. During their transport to the plasma membrane, the distance between Gag and gRNA decreases together with an increase of gRNA packaging. Point mutations in the zinc finger patterns of the nucleocapsid domain of Gag caused an increase in the distance between Gag and gRNA as well as a sharp decrease of gRNA packaged into virions. Finally, we show that removal of stem loop 1 of the 5'-untranslated region does not interfere with gRNA packaging, whereas combined with the removal of stem loop 3 is sufficient to decrease but not abolish Gag-gRNA cluster formation and gRNA packaging. In conclusion, this morphometric analysis of Gag-gRNA cluster formation sheds new light on HIV-1 assembly that can be used to describe at nanoscale resolution other viral assembly steps involving RNA or protein-protein interactions.
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Productos del Gen gag , VIH-1 , Nucleoproteínas , Regiones no Traducidas 5' , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Genómica , VIH-1/genética , VIH-1/metabolismo , Microscopía Electrónica de Transmisión , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , ARN Guía de Kinetoplastida , ARN Viral/genética , ARN Viral/metabolismo , Ensamble de Virus/genéticaRESUMEN
Hepatitis C is a major threat to public health for which an effective treatment is available, but a prophylactic vaccine is still needed to control this disease. We designed a vaccine based on chimeric HBV-HCV envelope proteins forming subviral particles (SVPs) that induce neutralizing antibodies against HCV in vitro. Here, we aimed to increase the neutralizing potential of those antibodies, by using HBV-HCV SVPs bearing apolipoprotein E (apoE). These particles were produced by cultured stable mammalian cell clones, purified and characterized. We found that apoE was able to interact with both chimeric HBV-HCV (E1-S and E2-S) proteins, and with the wild-type HBV S protein. ApoE was also detected on the surface of purified SVPs and improved the folding of HCV envelope proteins, but its presence lowered the incorporation of E2-S protein. Immunization of New Zealand rabbits resulted in similar anti-S responses for all rabbits, whereas anti-E1/-E2 antibody titers varied according to the presence or absence of apoE. Regarding the neutralizing potential of these anti-E1/-E2 antibodies, it was higher in rabbits immunized with apoE-bearing particles. In conclusion, the association of apoE with HCV envelope proteins may be a good strategy for improving HCV vaccines based on viral envelope proteins.
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Apolipoproteínas E/administración & dosificación , Apolipoproteínas E/inmunología , Hepacivirus/inmunología , Virus de la Hepatitis B/inmunología , Vacunas contra Hepatitis Viral/administración & dosificación , Vacunas contra Hepatitis Viral/inmunología , Animales , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Neutralizantes/sangre , Presentación de Antígeno/inmunología , Línea Celular , Femenino , Hepatitis C/inmunología , Hepatitis C/prevención & control , Anticuerpos contra la Hepatitis C/biosíntesis , Anticuerpos contra la Hepatitis C/sangre , Humanos , Evasión Inmune , Conejos , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/inmunología , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
INTRODUCTION: The unique anatomical characteristics of the forearm bones makes their reconstruction challenging. The aim of this study was to report the surgical methods and results of the induced membrane technique applied to traumatic forearm bone defects. MATERIAL AND METHODS: We evaluated retrospectively a case series of 13 patients operated between 2010 and 2017. The first surgical step consisted of debridement of the fracture site and implantation of a cement spacer with appropriate fixation. The anatomy of the forearm skeleton had to be restored. The second step, done 6 weeks later, consisted of removing the cement spacer and applying cancellous bone autograft harvested from the iliac crest. The outcome measures were radiological bone union, need for surgical revision, and postoperative wrist range of motion. RESULTS: All 13 patients were men, with a mean age of 39 years (18-67). The average follow-up was 2.5 years. Eleven patients were suffering from a nonunion and two from a post-traumatic bone defect. Six patients had an identified preoperative infection. Three patients had previously undergone an unsuccessful treatment for their nonunion with bone addition. The maximum length of bone reconstruction was 12 cm. Union was achieved in 12 of 13 patients in a mean of 5 months (3-8). The other patient died during the postoperative course. Two patients needed revision surgery: ulnar shortening osteotomy (1 case) and additional tendon reconstruction (1 case). The mean pronosupination range was 123° on average (55-180°). The mean flexion-extension range was 106° (90-130°). CONCLUSION: The induced membrane technique is a reliable reconstruction technique that is well suited to reconstruction of the forearm skeleton. LEVEL OF EVIDENCE: IV, retrospective study.
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Antebrazo , Fracturas no Consolidadas , Adulto , Trasplante Óseo/métodos , Fracturas no Consolidadas/cirugía , Humanos , Masculino , Rango del Movimiento Articular , Estudios Retrospectivos , Resultado del Tratamiento , Articulación de la MuñecaRESUMEN
The human APOBEC3A (A3A) cytidine deaminase is a powerful DNA mutator enzyme recognized as a major source of somatic mutations in tumor cell genomes. However, there is a discrepancy between APOBEC3A mRNA levels after interferon stimulation in myeloid cells and A3A detection at the protein level. To understand this difference, we investigated the expression of two novel alternative "A3Alt" proteins encoded in the +1-shifted reading frame of the APOBEC3A gene. A3Alt-L and its shorter isoform A3Alt-S appear to be transmembrane proteins targeted to the mitochondrial compartment that induce membrane depolarization and apoptosis. Thus, the APOBEC3A gene represents a new example wherein a single gene encodes two proapoptotic proteins, A3A cytidine deaminases that target the genome and A3Alt proteins that target mitochondria.
Asunto(s)
Citidina Desaminasa/genética , Citidina Desaminasa/fisiología , Mitocondrias/genética , Proteínas/genética , Proteínas/fisiología , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Citidina Desaminasa/metabolismo , ADN/genética , Mutación del Sistema de Lectura/genética , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Genoma/genética , Humanos , Mitocondrias/metabolismo , Mutación/genética , Proteínas/metabolismo , ARN Mensajero/genética , Sistemas de Lectura/genéticaRESUMEN
CC17 Streptococcus agalactiae carrying group-A prophages is increasingly responsible for neonatal infections. To investigate the impact of the genetic features of a group-A prophage, we first conducted an in silico analysis of the genome of 12/111phiA, a group-A prophage carried by a strain responsible for a bloodstream infection in a parturient. This revealed a Restriction Modification system, suggesting a prophage maintenance strategy and five ORFs of interest for the host and encoding a type II toxin antitoxin system RelB/YafQ, an endonuclease, an S-adenosylmethionine synthetase MetK, and an StrP-like adhesin. Using the WT strain cured from 12/111phiA and constructing deleted mutants for the ORFs of interest, and their complemented mutants, we demonstrated an impact of prophage features on growth characteristics, cell morphology and biofilm formation. Our findings argue in favor of 12/111phiA domestication by the host and a role of prophage features in cell autoaggregation, glycocalyx and biofilm formation. We suggest that lysogeny may promote GBS adaptation to the acid environment of the vagina, consequently colonizing and infecting neonates.