24 results on '"João I. Mamede"'
Search Results
2. The SARS-CoV-2 S1 Spike Protein Promotes MAPK and NF-kB Activation in Human Lung Cells and Inflammatory Cytokine Production in Human Lung and Intestinal Epithelial Cells
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Christopher B. Forsyth, Lijuan Zhang, Abhinav Bhushan, Barbara Swanson, Li Zhang, João I. Mamede, Robin M. Voigt, Maliha Shaikh, Phillip A. Engen, and Ali Keshavarzian
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COVID-19 ,ARDS ,cytokine storm ,S1 spike protein ,SARS-CoV-2 ,PASC ,Biology (General) ,QH301-705.5 - Abstract
The coronavirus disease 2019 (COVID-19) pandemic began in January 2020 in Wuhan, China, with a new coronavirus designated SARS-CoV-2. The principal cause of death from COVID-19 disease quickly emerged as acute respiratory distress syndrome (ARDS). A key ARDS pathogenic mechanism is the “Cytokine Storm”, which is a dramatic increase in inflammatory cytokines in the blood. In the last two years of the pandemic, a new pathology has emerged in some COVID-19 survivors, in which a variety of long-term symptoms occur, a condition called post-acute sequelae of COVID-19 (PASC) or “Long COVID”. Therefore, there is an urgent need to better understand the mechanisms of the virus. The spike protein on the surface of the virus is composed of joined S1–S2 subunits. Upon S1 binding to the ACE2 receptor on human cells, the S1 subunit is cleaved and the S2 subunit mediates the entry of the virus. The S1 protein is then released into the blood, which might be one of the pivotal triggers for the initiation and/or perpetuation of the cytokine storm. In this study, we tested the hypothesis that the S1 spike protein is sufficient to activate inflammatory signaling and cytokine production, independent of the virus. Our data support a possible role for the S1 spike protein in the activation of inflammatory signaling and cytokine production in human lung and intestinal epithelial cells in culture. These data support a potential role for the SARS-CoV-2 S1 spike protein in COVID-19 pathogenesis and PASC.
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- 2022
- Full Text
- View/download PDF
3. HIV Infection and Spread between Th17 Cells
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Janet P. Zayas and João I. Mamede
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HIV-1 ,CD4+ T cells ,Th17 cells ,pathogenesis ,reservoirs ,cell-to-cell spread ,Microbiology ,QR1-502 - Abstract
HIV mainly targets CD4+ T cells, from which Th17 cells represent a major cell type, permissive, and are capable of supporting intracellular replication at mucosal sites. Th17 cells possess well-described dual roles, while being central to maintaining gut integrity, these may induce inflammation and contribute to autoimmune disorders; however, Th17 cells’ antiviral function in HIV infection is not completely understood. Th17 cells are star players to HIV-1 pathogenesis and a potential target to prevent or decrease HIV transmission. HIV-1 can be spread among permissive cells via direct cell-to-cell and/or cell-free infection. The debate on which mode of transmission is more efficient is still ongoing without a concrete conclusion yet. Most assessments of virus transmission analyzing either cell-to-cell or cell-free modes use in vitro systems; however, the actual interactions and conditions in vivo are not fully understood. The fact that infected breast milk, semen, and vaginal secretions contain a mix of both cell-free viral particles and infected cells presents an argument for the probability of HIV taking advantage of both modes of transmission to spread. Here, we review important insights and recent findings about the role of Th17 cells during HIV pathogenesis in mucosal surfaces, and the mechanisms of HIV-1 infection spread among T cells in tissues.
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- 2022
- Full Text
- View/download PDF
4. Sterol targeting drugs reveal life cycle stage-specific differences in trypanosome lipid rafts
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Aabha I. Sharma, Cheryl L. Olson, João I. Mamede, Felipe Gazos-Lopes, Conrad L. Epting, Igor C. Almeida, and David M. Engman
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Medicine ,Science - Abstract
Abstract Cilia play important roles in cell signaling, facilitated by the unique lipid environment of a ciliary membrane containing high concentrations of sterol-rich lipid rafts. The African trypanosome Trypanosoma brucei is a single-celled eukaryote with a single cilium/flagellum. We tested whether flagellar sterol enrichment results from selective flagellar partitioning of specific sterol species or from general enrichment of all sterols. While all sterols are enriched in the flagellum, cholesterol is especially enriched. T. brucei cycles between its mammalian host (bloodstream cell), in which it scavenges cholesterol, and its tsetse fly host (procyclic cell), in which it both scavenges cholesterol and synthesizes ergosterol. We wondered whether the insect and mammalian life cycle stages possess chemically different lipid rafts due to different sterol utilization. Treatment of bloodstream parasites with cholesterol-specific methyl-β-cyclodextrin disrupts both membrane liquid order and localization of a raft-associated ciliary membrane calcium sensor. Treatment with ergosterol-specific amphotericin B does not. The opposite results were observed with ergosterol-rich procyclic cells. Further, these agents have opposite effects on flagellar sterol enrichment and cell metabolism in the two life cycle stages. These findings illuminate differences in the lipid rafts of an organism employing life cycle-specific sterols and have implications for treatment.
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- 2017
- Full Text
- View/download PDF
5. A New Generation of Functional Tagged Proteins for HIV Fluorescence Imaging
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João I. Mamede, Joseph Griffin, Stéphanie Gambut, and Thomas J. Hope
- Subjects
fluorescent HIV ,integration competent tagged viruses ,HIV early-steps ,Microbiology ,QR1-502 - Abstract
During the last decade, there was a marked increase in the development of tools and techniques to study the molecular mechanisms of the HIV replication cycle by using fluorescence microscopy. Researchers often apply the fusion of tags and fluorophores to viral proteins, surrogate proteins, or dyes to follow individual virus particles while they progress throughout infection. The inclusion of such fusion motifs or surrogates frequently disrupts viral infectivity or results in a change of the wild-type phenotype. Here, we detail the construction and functional characterization of two new constructs where we fused fluorescent proteins to the N-terminus of HIV-1 Integrase. In the first, IN is recruited into assembling particles via a codon optimized Gag to complement other viral constructs, while the second is fused to a Gag-Pol expression vector fully capable of integration. Our data shows that N-terminal tagged IN is functional for integration by both recovery of integration of catalytically inactive IN and by the successful infectivity of viruses carrying only labeled IN. These tools will be important to study the individual behavior of viral particles and associate such behavior to infectivity.
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- 2021
- Full Text
- View/download PDF
6. Restriction factor compendium for influenza A virus reveals a mechanism for evasion of autophagy
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Courtney Nguyen, Danielle L. Swaney, Lars Pache, Hong M. Moulton, David A. Stein, Shashank Tripathi, Dexter Pratt, Trey Ideker, Stephen Soonthornvacharin, David Jimenez-Morales, Maite Sanchez-Aparicio, Nish Beltran-Raygoza, Paul D. De Jesus, Randy A. Albrecht, Kelsey M. Haas, Adolfo García-Sastre, Judd F. Hultquist, João I. Mamede, Guojun Wang, Laura Martin-Sancho, Ariel Rodriguez-Frandsen, Christopher Churas, Max W. Chang, Sara Brin Rosenthal, Thong T. Nguyen, Nevan J. Krogan, Sumit K. Chanda, Michael J. McGregor, Laura Riva, and Christopher Benner
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Microbiology (medical) ,Immunology ,Regulator ,Biology ,Virus Replication ,medicine.disease_cause ,Proteomics ,Antiviral Agents ,Applied Microbiology and Biotechnology ,Microbiology ,Article ,Viral Matrix Proteins ,Vaccine Related ,Cell membrane ,Transcriptome ,Biodefense ,Autophagy ,Genetics ,Influenza A virus ,medicine ,Humans ,2.2 Factors relating to the physical environment ,2.1 Biological and endogenous factors ,Aetiology ,Immune Evasion ,Prevention ,GTPase-Activating Proteins ,rab7 GTP-Binding Proteins ,Cell Biology ,Influenza ,In vitro ,Cell biology ,Infectious Diseases ,Emerging Infectious Diseases ,medicine.anatomical_structure ,rab GTP-Binding Proteins ,Medical Microbiology ,Cytoplasm ,Host-Pathogen Interactions ,Pneumonia & Influenza ,Lysosomes ,Infection ,Protein Binding - Abstract
The fate of influenza A virus (IAV) infection in the host cell depends on the balance between cellular defence mechanisms and viral evasion strategies. To illuminate the landscape of IAV cellular restriction, we generated and integrated global genetic loss-of-function screens with transcriptomics and proteomics data. Our multi-omics analysis revealed a subset of both IFN-dependent and independent cellular defence mechanisms that inhibit IAV replication. Amongst these, the autophagy regulator TBC1 domain family member 5 (TBC1D5), which binds Rab7 to enable fusion of autophagosomes and lysosomes, was found to control IAV replication in vitro and in vivo and to promote lysosomal targeting of IAV M2 protein. Notably, IAV M2 was observed to abrogate TBC1D5-Rab7 binding through a physical interaction with TBC1D5 via its cytoplasmic tail. Our results provide evidence for the molecular mechanism utilised by IAV M2 protein to escape lysosomal degradation and traffic to the cell membrane, where it supports IAV budding and growth.
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- 2021
7. De novo emergence of SARS‐CoV‐2 spike mutations in immunosuppressed patients
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Lacy M. Simons, Egon A. Ozer, Stephanie Gambut, Taylor J. Dean, Li Zhang, Pavan Bhimalli, Jeffrey R. Schneider, João I. Mamede, Michael G. Ison, Reem Karmali, Leo I. Gordon, Ramon Lorenzo‐Redondo, and Judd F. Hultquist
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Immunocompromised Host ,Transplantation ,Infectious Diseases ,SARS-CoV-2 ,Mutation ,Humans ,COVID-19 ,Antibodies, Viral ,Antiviral Agents ,Antibodies, Neutralizing - Abstract
The continuing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with decreased susceptibility to neutralizing antibodies is of clinical importance. Several spike mutations associated with immune escape have evolved independently in association with different variants of concern (VOCs). How and when these mutations arise is still unclear. We hypothesized that such mutations might arise in the context of persistent viral replication in immunosuppressed hosts.Nasopharyngeal specimens were collected longitudinally from two immunosuppressed patients with persistent SARS-CoV-2 infection. Plasma was collected from these same patients late in disease course. SARS-CoV-2 whole genome sequencing was performed to assess the emergence and frequency of mutations over time. Select Spike mutations were assessed for their impact on viral entry and antibody neutralization in vitro.Our sequencing results revealed the intrahost emergence of spike mutations that are associated with circulating VOCs in both immunosuppressed patients (del241-243 and E484Q in one patient, and E484K in the other). These mutations decreased antibody-mediated neutralization of pseudotyped virus particles in cell culture, but also decreased efficiency of spike-mediated cell entry.These observations demonstrate the de novo emergence of SARS-CoV-2 spike mutations with enhanced immune evasion in immunosuppressed patients with persistent infection. These data suggest one potential mechanism for the evolution of VOCs and emphasize the importance of continued efforts to develop antiviral drugs for suppression of viral replication in hospitalized settings.
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- 2022
8. Bulk IgG glycosylation predicts COVID-19 severity and vaccine antibody response
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Michelle K. Ash, Pavan P. Bhimalli, Byoung-Kyu Cho, Basil Baby Mattamana, Stéphanie Gambut, Imad Tarhoni, Cristina L. Fhied, Anjelica F. Reyes, Samantha J. Welninski, Jaison Arivalagan, Fernanda Negrão, Renu Goel, Todd L. Beck, Thomas J. Hope, Beverly E. Sha, Young Ah Goo, Lena Al-Harthi, João I. Mamede, Jeffrey A. Borgia, Neil L. Kelleher, and Jeffrey R. Schneider
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General Biochemistry, Genetics and Molecular Biology - Abstract
Although vaccination efforts have expanded, there are still gaps in our understanding surrounding the immune response to SARS-CoV-2. Measuring IgG Fc glycosylation provides insight into an infected individual's inflammatory state, among other functions. We set out to interrogate bulk IgG glycosylation changes from SARS-CoV-2 infection and vaccination, using plasma from mild or hospitalized COVID-19 patients, and from vaccinated individuals. Inflammatory glycans are elevated in hospitalized COVID-19 patients and increase over time, while mild patients have anti-inflammatory glycans that increase over time, including increased sialic acid correlating with RBD antibody levels. Vaccinated individuals with low RBD antibody levels and low neutralization have the same IgG glycan traits as hospitalized COVID-19 patients. In addition, a small vaccinated cohort reveals a decrease in inflammatory glycans associated with peak IgG concentrations and neutralization. This report characterizes the bulk IgG glycome associated with COVID-19 severity and vaccine responsiveness and can help guide future studies into SARS-CoV-2 protective immunity.
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- 2022
9. Recognition of HIV-1 Capsid Licenses Innate Immune Response to Viral Infection
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Sunnie M Yoh, João I. Mamede, Derrick Lau, Narae Ahn, Maria T Sánchez-Aparicio, Joshua Temple, Andrew Tuckwell, Nina V. Fuchs, Gianguido C. Cianci, Laura Riva, Heather Curry, Xin Yin, Stéphanie Gambut, Lacy M. Simons, Judd F. Hultquist, Renate König, Yong Xiong, Adolfo García-Sastre, Till Böcking, Thomas J. Hope, and Sumit K. Chanda
- Abstract
SUMMARYCyclic GMP-AMP synthase (cGAS) is a primary sensor of aberrant DNA that governs an innate immune signaling cascade, leading to the induction of the type-I interferon response. We have previously identified polyglutamine binding protein 1, PQBP1, as an adaptor molecule required for cGAS-mediated innate immune response of lentiviruses, including the human immunodeficiency virus 1 (HIV-1), but dispensable for the recognition of DNA viruses. HIV-1- encoded DNA is synthesized as a single copy from its RNA genome, and is subsequently integrated into the host chromatin. HIV-1 then produces progeny through amplification and packaging of its RNA genome, thus, in contrast to DNA viruses, HIV-1 DNA is both transient and of low abundance. However, the molecular basis for the detection and verification of this low abundance HIV-1 DNA pathogen-associated molecular pattern (PAMP) is not understood. Here, we elucidate a two-factor authentication strategy that is employed by the innate immune surveillance machinery to selectively respond to the low concentration of PAMP, while discerning these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates intact viral capsid, which serves as a primary verification step for the viral nucleic acid cargo. As the reverse transcription and capsid disassembly initiate, cGAS protein is then recruited to the capsid in a PQBP1-dependent manner, enabling cGAS molecules to be co-positioned at the site of PAMP generation. Thus, these data indicate that PQBP1 recognition of the HIV-1 capsid sanctions a robust cGAS-dependent response to a limited abundance and short-lived DNA PAMP. Critically, this illuminates a molecular strategy wherein the modular recruitment of co-factors to germline encoded pattern recognition receptors (PRRs) serves to enhance repertoire of pathogens that can be sensed by the innate immune surveillance machinery.
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- 2022
10. HIV Infection and Spread between Th
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Janet P, Zayas and João I, Mamede
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Disease Models, Animal ,Mucous Membrane ,HIV-1 ,Animals ,Humans ,Th17 Cells ,HIV Infections ,Virus Replication - Abstract
HIV mainly targets CD4
- Published
- 2021
11. Multiple expansions of globally uncommon SARS-CoV-2 lineages in Nigeria
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Egon A, Ozer, Lacy M, Simons, Olubusuyi M, Adewumi, Adeola A, Fowotade, Ewean C, Omoruyi, Johnson A, Adeniji, Oluseyi A, Olayinka, Taylor J, Dean, Janet, Zayas, Pavan P, Bhimalli, Michelle K, Ash, Almoustapha I, Maiga, Anou M, Somboro, Mamoudou, Maiga, Adam, Godzik, Jeffrey R, Schneider, João I, Mamede, Babafemi O, Taiwo, Judd F, Hultquist, and Ramon, Lorenzo-Redondo
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Adult ,Aged, 80 and over ,Male ,Adolescent ,SARS-CoV-2 ,COVID-19 ,Nigeria ,Genome, Viral ,Middle Aged ,Antibodies, Viral ,Antibodies, Neutralizing ,Africa, Western ,Young Adult ,Child, Preschool ,Mutation ,Spike Glycoprotein, Coronavirus ,Humans ,Female ,Child ,Phylogeny ,Aged - Abstract
Disparities in SARS-CoV-2 genomic surveillance have limited our understanding of the viral population dynamics and may delay identification of globally important variants. Despite being the most populated country in Africa, Nigeria has remained critically under sampled. Here, we report sequences from 378 SARS-CoV-2 isolates collected in Oyo State, Nigeria between July 2020 and August 2021. In early 2021, most isolates belonged to the Alpha "variant of concern" (VOC) or the Eta lineage. Eta outcompeted Alpha in Nigeria and across West Africa, persisting in the region even after expansion of an otherwise rare Delta sub-lineage. Spike protein from the Eta variant conferred increased infectivity and decreased neutralization by convalescent sera in vitro. Phylodynamic reconstructions suggest that Eta originated in West Africa before spreading globally and represented a VOC in early 2021. These results demonstrate a distinct distribution of SARS-CoV-2 lineages in Nigeria, and emphasize the need for improved genomic surveillance worldwide.
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- 2021
12. Recognition of HIV-1 capsid by PQBP1 licenses an innate immune sensing of nascent HIV-1 DNA
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Sunnie M. Yoh, João I. Mamede, Derrick Lau, Narae Ahn, Maria T. Sánchez-Aparicio, Joshua Temple, Andrew Tuckwell, Nina V. Fuchs, Gianguido C. Cianci, Laura Riva, Heather Curry, Xin Yin, Stéphanie Gambut, Lacy M. Simons, Judd F. Hultquist, Renate König, Yong Xiong, Adolfo García-Sastre, Till Böcking, Thomas J. Hope, and Sumit K. Chanda
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DNA-Binding Proteins ,Capsid ,Pathogen-Associated Molecular Pattern Molecules ,HIV-1 ,Humans ,Cell Biology ,DNA ,Molecular Biology ,Nucleotidyltransferases ,Immunity, Innate - Abstract
We have previously described polyglutamine-binding protein 1 (PQBP1) as an adapter required for the cyclic GMP-AMP synthase (cGAS)-mediated innate response to the human immunodeficiency virus 1 (HIV-1) and other lentiviruses. Cytoplasmic HIV-1 DNA is a transient and low-abundance pathogen-associated molecular pattern (PAMP), and the mechanism for its detection and verification is not fully understood. Here, we show a two-factor authentication strategy by the innate surveillance machinery to selectively respond to the low concentration of HIV-1 DNA, while distinguishing these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates the intact viral capsid, and this serves as a primary verification step for the viral nucleic acid cargo. As reverse transcription and capsid disassembly initiate, cGAS is recruited to the capsid in a PQBP1-dependent manner. This positions cGAS at the site of PAMP generation and sanctions its response to a low-abundance DNA PAMP.
- Published
- 2021
13. Coincident rapid expansion of two SARS-CoV-2 lineages with enhanced infectivity in Nigeria
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Egon A. Ozer, Michelle K Ash, Babafemi Taiwo, Ramon Lorenzo-Redondo, Adeola Fowotade, Judd F. Hultquist, Ewean Chukwuma Omoruyi, O. M. Adewumi, Pavan P. Bhimalli, Lacy M Simons, Johnson Adekunle Adeniji, Jeffrey R Schneider, Taylor J Dean, Adam Godzik, Janet Zayas, and João I. Mamede
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education.field_of_study ,Lineage (genetic) ,Phylogenetic tree ,Evolutionary biology ,Phylogenetics ,Transmission (medicine) ,Population ,Pandemic ,Global health ,Outbreak ,Biology ,education - Abstract
The spread of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), has resulted in a global pandemic that has claimed the lives of millions of people. Genomic surveillance of the virus has proven to be a critical tool for tracking the emergence and spread of variants with increased transmission or immune evasion potential. Despite the global distribution of infection, differences in viral genomic surveillance capabilities between countries and regions have resulted in gaps in our understanding of the viral population dynamics underlying the pandemic. Nigeria, despite having the largest population of any country in Africa, has had relatively little SARS-CoV-2 sequence data made publicly available. In this study, we report the whole-genome sequences of 74 SARS-CoV-2 isolates collected from individuals in Oyo State, Nigeria over the first two weeks of January 2021. Forty-six of the isolates belong to the B.1.1.7 "UK variant" lineage. Comparison to available regional and global sequences suggest that the B.1.1.7 isolates in Nigeria are primarily monophyletic, possibly representing a singular successful introduction into the country. The majority of the remaining isolates (17 of 74) belong to the B.1.525 lineage, which contains multiple spike protein mutations, including the E484K mutation associated with potential immune escape. Indeed, Nigeria has the highest reported frequency of this lineage despite its relative rarity worldwide. Phylogenetic analysis of the B.1.525 isolates in this study relative to other local and global isolates suggested a recent origin and rapid expansion of this lineage in Nigeria, with the country serving as a potential source for this lineage in other outbreaks. These results demonstrate the importance of genomic surveillance for identifying SARS-CoV-2 variants of concern in Nigeria and in other undersampled regions across the globe.
- Published
- 2021
14. A New Generation of Functional Tagged Proteins for HIV Fluorescence Imaging
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Joseph Griffin, Thomas J. Hope, Stéphanie Gambut, and João I. Mamede
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0301 basic medicine ,Fluorescence-lifetime imaging microscopy ,viruses ,030106 microbiology ,lcsh:QR1-502 ,Human immunodeficiency virus (HIV) ,HIV Infections ,Computational biology ,medicine.disease_cause ,Virus Replication ,lcsh:Microbiology ,Virus ,Fluorescence ,Article ,Cell Line ,03 medical and health sciences ,Viral Proteins ,Virology ,Cell Line, Tumor ,Fluorescence microscope ,medicine ,Humans ,Infectivity ,Expression vector ,fluorescent HIV ,HIV early-steps ,biology ,Integrases ,Chemistry ,integration competent tagged viruses ,Optical Imaging ,Virion ,Phenotype ,Integrase ,030104 developmental biology ,Infectious Diseases ,HEK293 Cells ,biology.protein ,HIV-1 ,HeLa Cells - Abstract
During the last decade, there was a marked increase in the development of tools and techniques to study the molecular mechanisms of the HIV replication cycle by using fluorescence microscopy. Researchers often apply the fusion of tags and fluorophores to viral proteins, surrogate proteins, or dyes to follow individual virus particles while they progress throughout infection. The inclusion of such fusion motifs or surrogates frequently disrupts viral infectivity or results in a change of the wild-type phenotype. Here, we detail the construction and functional characterization of two new constructs where we fused fluorescent proteins to the N-terminus of HIV-1 Integrase. In the first, IN is recruited into assembling particles via a codon optimized Gag to complement other viral constructs, while the second is fused to a Gag-Pol expression vector fully capable of integration. Our data shows that N-terminal tagged IN is functional for integration by both recovery of integration of catalytically inactive IN and by the successful infectivity of viruses carrying only labeled IN. These tools will be important to study the individual behavior of viral particles and associate such behavior to infectivity.
- Published
- 2021
15. Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells
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Grant J. Jensen, Zachary Freyberg, Zachary J. Farino, Shrawan Kumar Mageswaran, Stephen D. Carter, João I. Mamede, Thomas J. Hope, and Catherine M. Oikonomou
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In situ ,0301 basic medicine ,Fluorophore ,Biology ,Signal ,Article ,Fluorescence ,law.invention ,chemistry.chemical_compound ,03 medical and health sciences ,Structural Biology ,law ,Cell Line, Tumor ,Insulin-Secreting Cells ,Fluorescence microscope ,Animals ,Humans ,030304 developmental biology ,Luminescent Proteins ,0303 health sciences ,Chemistry ,Secretory Vesicles ,030302 biochemistry & molecular biology ,Resolution (electron density) ,Cryoelectron Microscopy ,Fibroblasts ,Secretory Vesicle ,Cell biology ,Mitochondria ,Rats ,Autofluorescence ,030104 developmental biology ,Microscopy, Fluorescence ,Ultrastructure ,Biophysics ,Target protein ,Electron microscope ,Fluorescein-5-isothiocyanate ,HeLa Cells - Abstract
Cryogenic correlated light and electron microscopy (cryo-CLEM) is a valuable tool for studying biological processesin situ. In cryo-CLEM, a target protein of interest is tagged with a fluorophore and the location of the corresponding fluorescent signal is used to identify the structure in low-contrast but feature-rich cryo-EM images. To date, cryo-CLEM studies of mammalian cells have relied on very bright organic dyes or fluorescent protein tags concentrated in virus particles. Here we describe a method to expand the application of cryo-CLEM to cells harboring genetically-encoded fluorescent proteins. We discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80K). Compared to fluorescent protein tags, these sources of autofluorescence exhibit a broader spectrum of fluorescence, which we exploited to develop a simple, robust approach to discriminate between the two. We validate this method in INS-1 E cells using a mitochondrial marker, and apply it to study the ultrastructural variability of secretory granules in a near-native state within intact INS-1E pancreatic cells by high-resolution 3D electron cryotomography.
- Published
- 2017
16. Early cytoplasmic uncoating is associated with infectivity of HIV-1
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Gianguido C. Cianci, João I. Mamede, Thomas J. Hope, and Meegan R. Anderson
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0301 basic medicine ,Cytoplasm ,viruses ,Cell ,HIV Infections ,Biology ,Virus Replication ,03 medical and health sciences ,chemistry.chemical_compound ,Capsid ,Live cell imaging ,Virus Uncoating ,medicine ,Humans ,Infectivity ,Multidisciplinary ,Virology ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,PNAS Plus ,Viral replication ,chemistry ,Host-Pathogen Interactions ,HIV-1 ,Capsid Proteins ,DNA - Abstract
After fusion, HIV delivers its conical capsid into the cytoplasm. To release the contained reverse-transcribing viral genome, the capsid must disassemble in a process termed uncoating. Defining the kinetics, dynamics, and cellular location of uncoating of virions leading to infection has been confounded by defective, noninfectious particles and the stochastic minefield blocking access to host DNA. We used live-cell fluorescent imaging of intravirion fluid phase markers to monitor HIV-1 uncoating at the individual particle level. We find that HIV-1 uncoating of particles leading to infection is a cytoplasmic process that occurs ∼30 min postfusion. Most, but not all, of the capsid protein is rapidly shed in tissue culture and primary target cells, independent of entry pathway. Extended time-lapse imaging with less than one virion per cell allows identification of infected cells by Gag-GFP expression and directly links individual particle behavior to infectivity, providing unprecedented insights into the biology of HIV infection.
- Published
- 2017
17. Sterol targeting drugs reveal life cycle stage-specific differences in trypanosome lipid rafts
- Author
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David M. Engman, Felipe Gazos-Lopes, João I. Mamede, Aabha I. Sharma, Cheryl L. Olson, Igor C. Almeida, and Conrad L. Epting
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0301 basic medicine ,Tsetse Flies ,Science ,Cell ,Trypanosoma brucei brucei ,Flagellum ,Trypanosoma brucei ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Membrane Microdomains ,Amphotericin B ,Ergosterol ,medicine ,polycyclic compounds ,Animals ,Ciliary membrane ,Lipid raft ,Life Cycle Stages ,Multidisciplinary ,biology ,Cholesterol ,beta-Cyclodextrins ,biology.organism_classification ,Sterol ,Cell biology ,Sterols ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,Biochemistry ,Flagella ,Medicine ,lipids (amino acids, peptides, and proteins) - Abstract
Cilia play important roles in cell signaling, facilitated by the unique lipid environment of a ciliary membrane containing high concentrations of sterol-rich lipid rafts. The African trypanosome Trypanosoma brucei is a single-celled eukaryote with a single cilium/flagellum. We tested whether flagellar sterol enrichment results from selective flagellar partitioning of specific sterol species or from general enrichment of all sterols. While all sterols are enriched in the flagellum, cholesterol is especially enriched. T. brucei cycles between its mammalian host (bloodstream cell), in which it scavenges cholesterol, and its tsetse fly host (procyclic cell), in which it both scavenges cholesterol and synthesizes ergosterol. We wondered whether the insect and mammalian life cycle stages possess chemically different lipid rafts due to different sterol utilization. Treatment of bloodstream parasites with cholesterol-specific methyl-β-cyclodextrin disrupts both membrane liquid order and localization of a raft-associated ciliary membrane calcium sensor. Treatment with ergosterol-specific amphotericin B does not. The opposite results were observed with ergosterol-rich procyclic cells. Further, these agents have opposite effects on flagellar sterol enrichment and cell metabolism in the two life cycle stages. These findings illuminate differences in the lipid rafts of an organism employing life cycle-specific sterols and have implications for treatment.
- Published
- 2017
18. PI-4 Trogocytosis of viral immune complexes in the context of HIV-1 infection
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Chiara Orlandi, Danijela Maric, Yi Hu, Robin Flinko, Thomas J. Hope, João I. Mamede, George K. Lewis, and Krishanu Ray
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Infectious Diseases ,Immune system ,Trogocytosis ,Immunology ,Human immunodeficiency virus (HIV) ,medicine ,Pharmacology (medical) ,Context (language use) ,Biology ,medicine.disease_cause - Published
- 2019
19. KIF5B and Nup358 Cooperatively Mediate the Nuclear Import of HIV-1 during Infection
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Adarsh Dharan, Edward M. Campbell, Matthias Majetschak, Abhishek Tripathi, João I. Mamede, Thomas J. Hope, and Sarah Talley
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0301 basic medicine ,RNA viruses ,Cytoplasm ,Cultured tumor cells ,Kinesins ,HIV Infections ,Pathology and Laboratory Medicine ,Biochemistry ,Viral Packaging ,Immunodeficiency Viruses ,Image Processing, Computer-Assisted ,Medicine and Health Sciences ,Small interfering RNAs ,Nuclear pore ,lcsh:QH301-705.5 ,Staining ,3. Good health ,Cell biology ,Nucleic acids ,medicine.anatomical_structure ,Capsid ,Medical Microbiology ,Cell Processes ,Gene Knockdown Techniques ,Viral Pathogens ,Viruses ,Cell lines ,Pathogens ,Cellular Structures and Organelles ,Biological cultures ,Research Article ,lcsh:Immunologic diseases. Allergy ,Immunology ,Dynein ,Blotting, Western ,Active Transport, Cell Nucleus ,Biology ,Viral Structure ,Real-Time Polymerase Chain Reaction ,Microbiology ,03 medical and health sciences ,Cyclophilin A ,Viral entry ,Virology ,Retroviruses ,medicine ,Viral Core ,Genetics ,Humans ,Nuclear Import ,HeLa cells ,Non-coding RNA ,Molecular Biology ,Microbial Pathogens ,Cytoplasmic Staining ,Cell Nucleus ,Lentivirus ,Organisms ,Biology and Life Sciences ,HIV ,Cell Biology ,Cell cultures ,Molecular biology ,Viral Replication ,Gene regulation ,Nuclear Pore Complex Proteins ,Research and analysis methods ,Cell nucleus ,030104 developmental biology ,HEK293 Cells ,lcsh:Biology (General) ,Microscopy, Fluorescence ,Specimen Preparation and Treatment ,HIV-1 ,RNA ,Parasitology ,Gene expression ,Nuclear transport ,lcsh:RC581-607 ,Molecular Chaperones - Abstract
Following envelope mediated fusion, the HIV-1 core is released into the cytoplasm of the target cell and undergoes a series of trafficking and replicative steps that result in the nuclear import of the viral genome, which ultimately leads to the integration of the proviral DNA into the host cell genome. Previous studies have found that disruption of microtubules, or depletion of dynein or kinesin motors, perturb the normal uncoating and trafficking of the viral genome. Here, we show that the Kinesin-1 motor, KIF5B, induces a relocalization of the nuclear pore component Nup358 into the cytoplasm during HIV-1 infection. This relocalization of NUP358 is dependent on HIV-1 capsid, and NUP358 directly associates with viral cores following cytoplasmic translocation. This interaction between NUP358 and the HIV-1 core is dependent on multiple capsid binding surfaces, as this association is not observed following infection with capsid mutants in which a conserved hydrophobic binding pocket (N74D) or the cyclophilin A binding loop (P90A) is disrupted. KIF5B knockdown also prevents the nuclear entry and infection by HIV-1, but does not exert a similar effect on the N74D or P90A capsid mutants which do not rely on Nup358 for nuclear import. Finally, we observe that the relocalization of Nup358 in response to CA is dependent on cleavage protein and polyadenylation factor 6 (CPSF6), but independent of cyclophilin A. Collectively, these observations identify a previously unappreciated role for KIF5B in mediating the Nup358 dependent nuclear import of the viral genome during infection., Author Summary Fusion of viral and target cell membranes releases the HIV-1 viral capsid, which houses the viral RNA and proteins necessary for viral reverse transcription and integration, into the cytoplasm of target cells. To complete infection, the viral capsid must ultimately traffic to the nucleus and undergo a process known as uncoating to allow the nuclear import of the viral genome into the nucleus, where it subsequently integrates into the genome of the target cell. Here, we show that the concerted actions of microtubule motor KIF5B and the nuclear pore component Nup358 cooperatively facilitate the uncoating and nuclear import of the viral genome. Moreover, we also identify the determinants in the viral capsid protein, which forms the viral capsid core, that are required for KIF5B dependent nuclear entry. These studies reveal a novel role for the microtubule motor KIF5B in the nuclear import of the viral genome and reveal potential intervention targets for therapeutic intervention.
- Published
- 2016
20. Detection and tracking of dual-labeled HIV particles using wide-field live cell imaging to follow viral core integrity
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Thomas J. Hope and João I. Mamede
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0301 basic medicine ,Materials science ,Human immunodeficiency virus (HIV) ,Cell Culture Techniques ,HIV Infections ,medicine.disease_cause ,Tracking (particle physics) ,Article ,Cell Line ,03 medical and health sciences ,Live cell imaging ,Virus Uncoating ,Microscopy ,medicine ,Fluorescence microscope ,Humans ,Staining and Labeling ,Viral core ,Optical Imaging ,Wide field ,Virology ,Cell biology ,030104 developmental biology ,HIV-1 ,Software - Abstract
Live cell imaging is a valuable technique that allows the characterization of the dynamic processes of the HIV-1 life cycle. Here, we present a method of production and imaging of dual-labeled HIV viral particles that allows the visualization of two events. Varying release of the intravirion fluid phase marker reveals virion fusion and the loss of the integrity of HIV viral cores with the use of live wide-field fluorescent microscopy.
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- 2016
21. Impact of Nucleoporin-Mediated Chromatin Localization and Nuclear Architecture on HIV Integration Site Selection
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João I. Mamede, Richard W. Wong, and Thomas J. Hope
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Virus Integration ,Immunology ,HIV integration ,Virus Attachment ,HIV Infections ,Biology ,Microbiology ,Genome ,Virology ,medicine ,Humans ,Gene ,Gems ,Genetics ,Cell Nucleus ,Chromatin ,Cell biology ,Nuclear Pore Complex Proteins ,Cell nucleus ,Microscopy, Electron ,medicine.anatomical_structure ,Viral replication ,Insect Science ,HIV-1 ,Nucleoporin ,HeLa Cells - Abstract
It has been known for a number of years that integration sites of human immunodeficiency virus type 1 (HIV-1) DNA show a preference for actively expressed chromosomal locations. A number of viral and cellular proteins are implicated in this process, but the underlying mechanism is not clear. Two recent breakthrough publications advance our understanding of HIV integration site selection by focusing on the localization of the preferred target genes of integration. These studies reveal that knockdown of certain nucleoporins and components of nucleocytoplasmic trafficking alter integration site preference, not by altering the trafficking of the viral genome but by altering the chromatin subtype localization relative to the structure of the nucleus. Here, we describe the link between the nuclear basket nucleoporins (Tpr and Nup153) and chromatin organization and how altering the host environment by manipulating nuclear structure may have important implications for the preferential integration of HIV into actively transcribed genes, facilitating efficient viral replication.
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- 2015
22. L’interaction de la cyclophiline A avec la capside lentivirale
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Valérie Courgnaud and João I. Mamede
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0303 health sciences ,03 medical and health sciences ,0302 clinical medicine ,Capsid ,General Medicine ,Biology ,Molecular biology ,030217 neurology & neurosurgery ,General Biochemistry, Genetics and Molecular Biology ,Virus ,030304 developmental biology - Published
- 2011
23. Glucose and Glutamine Metabolism Regulate Human Hematopoietic Stem Cell Lineage Specification
- Author
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Narla Mohandas, Dorota Klysz, Jawida Touhami, Marco Craveiro, João I. Mamede, Saverio Tardito, Sandrina Kinet, Stéphanie C. De Barros, Jean-Luc Battini, Xiuli An, Cédric Mongellaz, Naomi Taylor, Eyal Gottlieb, Peggy Merida, Vanessa Fritz, Marc Sitbon, Myriam Boyer-Clavel, Valérie S. Zimmermann, Leal Oburoglu, Gaspard Cretenet, Valérie Dardalhon, Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Cancer Research UK Beatson Institute [Glasgow], New York Blood Center, Zhengzhou University, BioCampus Montpellier (BCM), Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 1 (UM1)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)
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Regulation of gene expression ,0303 health sciences ,Lineage (genetic) ,Cellular differentiation ,Regulator ,Hematopoietic stem cell ,hemic and immune systems ,Cell Biology ,Biology ,Glutamine ,03 medical and health sciences ,Haematopoiesis ,0302 clinical medicine ,medicine.anatomical_structure ,Biochemistry ,hemic and lymphatic diseases ,medicine ,Genetics ,Molecular Medicine ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Stem cell ,030217 neurology & neurosurgery ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology - Abstract
SummaryThe metabolic state of quiescent hematopoietic stem cells (HSCs) is an important regulator of self-renewal, but it is unclear whether or how metabolic parameters contribute to HSC lineage specification and commitment. Here, we show that the commitment of human and murine HSCs to the erythroid lineage is dependent upon glutamine metabolism. HSCs require the ASCT2 glutamine transporter and active glutamine metabolism for erythroid specification. Blocking this pathway diverts EPO-stimulated HSCs to differentiate into myelomonocytic fates, altering in vivo HSC responses and erythroid commitment under stress conditions such as hemolytic anemia. Mechanistically, erythroid specification of HSCs requires glutamine-dependent de novo nucleotide biosynthesis. Exogenous nucleosides rescue erythroid commitment of human HSCs under conditions of limited glutamine catabolism, and glucose-stimulated nucleotide biosynthesis further enhances erythroid specification. Thus, the availability of glutamine and glucose to provide fuel for nucleotide biosynthesis regulates HSC lineage commitment under conditions of metabolic stress.
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24. Heterogeneous susceptibility of circulating SIV isolate capsids to HIV-interacting factors
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Jean-Luc Battini, Valérie Courgnaud, João I. Mamede, Marc Sitbon, Institut de Génétique Moléculaire de Montpellier (IGMM), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)
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Lentiviral capsids ,Pan troglodytes ,Virus Integration ,viruses ,Cypa ,Simian ,Virus Replication ,medicine.disease_cause ,Cell Line ,03 medical and health sciences ,TRIM5α ,Virology ,medicine ,Animals ,Humans ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Nup153 ,Immunodeficiency ,030304 developmental biology ,Infectivity ,0303 health sciences ,biology ,Research ,030302 biochemistry & molecular biology ,virus diseases ,Simian immunodeficiency virus ,biology.organism_classification ,medicine.disease ,Nup358/RanBP2 ,Phenotype ,3. Good health ,Infectious Diseases ,SIV ,Capsid ,Viral replication ,Host-Pathogen Interactions ,HIV-2 ,Immunology ,HIV-1 ,Capsid Proteins ,Simian Immunodeficiency Virus ,TRIMCyp - Abstract
Background Many species of non-human primates in Africa are naturally infected by simian immunodeficiency viruses (SIV) and humans stand at the forefront of exposure to these viruses in Sub-Saharan Africa. Cross-species transmission and adaptation of SIV to humans have given rise to human immunodeficiency viruses (HIV-1 and HIV-2) on twelve accountable, independent occasions. However, the determinants contributing to a simian-to-human lasting transmission are not fully understood. Following entry, viral cores are released into the cytoplasm and become the principal target of host cellular factors. Here, we evaluated cellular factors likely to be involved in potential new SIV cross-species transmissions. We investigated the interactions of capsids from naturally circulating SIV isolates with both HIV-1 restricting (i.e. TRIM5 proteins) and facilitating (i.e. cyclophilin A and nucleopore-associated Nup358/RanBP2 and Nup153) factors in single-round infectivity assays that reproduce early stages of the viral life-cycle. Results We show that human TRIM5α is unlikely to prevent cross-species transmission of any SIV we tested and observed that the SIV CA-CypA interaction is a widespread but not a universal feature. Moreover, entry in the nucleus of different SIV appeared to follow pathways that do not necessarily recruit Nup358/RanBP2 or Nup153, and this regardless of their interaction with CypA. Nevertheless, we found that, like HIV-1, human-adapted HIV-2 infection was dependent on Nup358/RanBP2 and Nup153 interactions for optimal infection. Furthermore, we found that, unlike HIV CA, SIV CA did not require a direct interaction with the Cyp-like domain of Nup358/RanBP2 to carry out successful infection. Conclusions Circulating SIV present a variety of phenotypes with regard to CA-interacting restricting or facilitating factors. Altogether, we unveiled unidentified pathways for SIV CA, which could also be exploited by HIV in different cellular contexts, to drive entry into the nucleus. Our findings warrant a closer evaluation of other potential defenses against circulating SIV.
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