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Gene expression is regulated by changes in chromatin architecture intrinsic to cellular differentiation and as an active response to environmental stimuli. Chromatin dynamics are a major driver of phenotypic diversity, regulation of development, and manifestation of disease. Remarkably, we know little about the evolutionary dynamics of chromatin reorganisation through time, data essential to characterise the impact of environmental stress during the ongoing biodiversity extinction crisis (20th-21st century). Linking the disparate fields of chromatin biology and museum science through their common use of the preservative formaldehyde (a constituent of formalin), we have generated historical chromatin profiles in museum specimens up to 117 years old. Historical chromatin profiles are reproducible, tissue-specific, sex-specific, and environmental condition-dependent in vertebrate specimens. Additionally, we show that over-fixation modulates differential chromatin accessibility to enable semi-quantitative estimates of relative gene expression in vertebrates and a yeast model. Our approach transforms formalin-fixed biological collections into an accurate, comprehensive, and global record of environmental impact on gene expression and phenotype.
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Cromatina , Formaldehído , Fijación del Tejido , Animales , Cromatina/metabolismo , Cromatina/genética , Fijación del Tejido/métodos , Femenino , Masculino , Vertebrados/genética , Fijadores/química , MuseosRESUMEN
Museum specimens represent an unparalleled record of historical genomic data. However, the widespread practice of formalin preservation has thus far impeded genomic analysis of a large proportion of specimens. Limited DNA sequencing from formalin-preserved specimens has yielded low genomic coverage with unpredictable success. We set out to refine sample processing methods and to identify specimen characteristics predictive of sequencing success. With a set of taxonomically diverse specimens collected between 1962 and 2006 and ranging in preservation quality, we compared the efficacy of several end-to-end whole genome sequencing workflows alongside a k-mer-based trimming-free read alignment approach to maximize mapping of endogenous sequence. We recovered complete mitochondrial genomes and up to 3× nuclear genome coverage from formalin-preserved tissues. Hot alkaline lysis coupled with phenol-chloroform extraction out-performed proteinase K digestion in recovering DNA, while library preparation method had little impact on sequencing success. The strongest predictor of DNA yield was overall specimen condition, which additively interacts with preservation conditions to accelerate DNA degradation. Here, we demonstrate a significant advance in capability beyond limited recovery of a small number of loci via PCR or target-capture sequencing. To facilitate strategic selection of suitable specimens for genomic sequencing, we present a decision-making framework that utilizes independent and nondestructive assessment criteria. Sequencing of formalin-preserved specimens will contribute to a greater understanding of temporal trends in genetic adaptation, including those associated with a changing climate. Our work enhances the value of museum collections worldwide by unlocking genomes of specimens that have been disregarded as a valid molecular resource.
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Formaldehído , Genoma Mitocondrial , ADN/genética , Preservación Biológica , Análisis de Secuencia de ADN/métodosRESUMEN
The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domains; previously C6orf106) was identified as a proviral factor for Hendra virus infection and was recently characterized to function as an inhibitor of type I interferon expression. Here, we have utilized transcriptome sequencing (RNA-seq) to define cellular pathways regulated by ILRUN in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of Caco-2 cells. We find that inhibition of ILRUN expression by RNA interference alters transcription profiles of numerous cellular pathways, including upregulation of the SARS-CoV-2 entry receptor ACE2 and several other members of the renin-angiotensin aldosterone system. In addition, transcripts of the SARS-CoV-2 coreceptors TMPRSS2 and CTSL were also upregulated. Inhibition of ILRUN also resulted in increased SARS-CoV-2 replication, while overexpression of ILRUN had the opposite effect, identifying ILRUN as a novel antiviral factor for SARS-CoV-2 replication. This represents, to our knowledge, the first report of ILRUN as a regulator of the renin-angiotensin-aldosterone system (RAAS). IMPORTANCE There is no doubt that the current rapid global spread of COVID-19 has had significant and far-reaching impacts on our health and economy and will continue to do so. Research in emerging infectious diseases, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is growing rapidly, with new breakthroughs in the understanding of host-virus interactions to assist with the development of innovative and exciting therapeutic strategies. Here, we present the first evidence that modulation of the human protein-coding gene ILRUN functions as an antiviral factor for SARS-CoV-2 infection, likely through its newly identified role in regulating the expression of SARS-CoV-2 entry receptors ACE2, TMPRSS2, and CTSL. These data improve our understanding of biological pathways that regulate host factors critical to SARS-CoV-2 infection, contributing to the development of antiviral strategies to deal with the current SARS-CoV-2 pandemic.
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Enzima Convertidora de Angiotensina 2/biosíntesis , COVID-19/metabolismo , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Proteínas de Neoplasias/metabolismo , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Animales , COVID-19/genética , Células CACO-2 , Catepsina L/biosíntesis , Catepsina L/genética , Chlorocebus aethiops , Humanos , Proteínas de Neoplasias/genética , Sistema Renina-Angiotensina , SARS-CoV-2/genética , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/genética , Células VeroRESUMEN
The global COVID-19 pandemic caused by SARS-CoV-2 has resulted in over 2.2 million deaths. Disease outcomes range from asymptomatic to severe with, so far, minimal genotypic change to the virus so understanding the host response is paramount. Transcriptomics has become incredibly important in understanding host-pathogen interactions; however, post-transcriptional regulation plays an important role in infection and immunity through translation and mRNA stability, allowing tight control over potent host responses by both the host and the invading virus. Here, we apply ribosome profiling to assess post-transcriptional regulation of host genes during SARS-CoV-2 infection of a human lung epithelial cell line (Calu-3). We have identified numerous transcription factors (JUN, ZBTB20, ATF3, HIVEP2 and EGR1) as well as select antiviral cytokine genes, namely IFNB1, IFNL1,2 and 3, IL-6 and CCL5, that are restricted at the post-transcriptional level by SARS-CoV-2 infection and discuss the impact this would have on the host response to infection. This early phase restriction of antiviral transcripts in the lungs may allow high viral load and consequent immune dysregulation typically seen in SARS-CoV-2 infection.
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Citocinas/genética , Procesamiento Postranscripcional del ARN , Ribosomas/metabolismo , Ribosomas/virología , SARS-CoV-2/inmunología , Factores de Transcripción/genética , Animales , Antivirales/antagonistas & inhibidores , Línea Celular Tumoral , Chlorocebus aethiops , Biología Computacional , Citocinas/metabolismo , Células Epiteliales/inmunología , Células Epiteliales/virología , Perfilación de la Expresión Génica , Interacciones Microbiota-Huesped , Humanos , Inmunidad Innata/genética , Pulmón/inmunología , Pulmón/virología , ARN Mensajero/metabolismo , RNA-Seq , Ribosomas/genética , SARS-CoV-2/metabolismo , Factores de Transcripción/metabolismo , Transcriptoma , Células VeroRESUMEN
The current pandemic has highlighted the ever-increasing risk of human to human spread of zoonotic pathogens. A number of medically-relevant zoonotic pathogens are negative-strand RNA viruses (NSVs). NSVs are derived from different virus families. Examples like Ebola are known for causing severe symptoms and high mortality rates. Some, like influenza, are known for their ease of person-to-person transmission and lack of pre-existing immunity, enabling rapid spread across many countries around the globe. Containment of outbreaks of NSVs can be difficult owing to their unpredictability and the absence of effective control measures, such as vaccines and antiviral therapeutics. In addition, there remains a lack of essential knowledge of the host-pathogen response that are induced by NSVs, particularly of the immune responses that provide protection. Vaccines are the most effective method for preventing infectious diseases. In fact, in the event of a pandemic, appropriate vaccine design and speed of vaccine supply is the most critical factor in protecting the population, as vaccination is the only sustainable defense. Vaccines need to be safe, efficient, and cost-effective, which is influenced by our understanding of the host-pathogen interface. Additionally, some of the major challenges of vaccines are the establishment of a long-lasting immunity offering cross protection to emerging strains. Although many NSVs are controlled through immunisations, for some, vaccine design has failed or efficacy has proven unreliable. The key behind designing a successful vaccine is understanding the host-pathogen interaction and the host immune response towards NSVs. In this paper, we review the recent research in vaccine design against NSVs and explore the immune responses induced by these viruses. The generation of a robust and integrated approach to development capability and vaccine manufacture can collaboratively support the management of outbreaking NSV disease health risks.
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The development of medical countermeasures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires robust viral assays. Here we have adapted a protocol for polyethylene glycol (PEG)-mediated precipitation of SARS-CoV-2 stocks without the need for ultracentrifugation. Virus precipitation resulted in a â¼1.5 log10 increase in SARS-CoV-2 titres of virus prepared in VeroE6 cells and enabled the infection of several immortalized human cell lines (Caco-2 and Calu-3) at a high multiplicity of infection not practically achievable without virus concentration. This protocol underscores the utility of PEG-mediated precipitation for SARS-CoV-2 and provides a resource for a range of coronavirus research areas.
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Betacoronavirus/aislamiento & purificación , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Polietilenglicoles/química , Animales , COVID-19 , Prueba de COVID-19 , Células CACO-2 , Chlorocebus aethiops , Infecciones por Coronavirus/diagnóstico , Humanos , Pandemias , Neumonía Viral/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2 , Ultracentrifugación/métodos , Células VeroRESUMEN
Regulation of type-I interferon (IFN) production is essential to the balance between antimicrobial defence and autoimmune disorders. The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domains, previously C6orf106) was recently characterised as an inhibitor of antiviral and proinflammatory cytokine (interferon-alpha/beta and tumor necrosis factor alpha) transcription. Currently there is a paucity of information about the molecular characteristics of ILRUN, despite it being associated with several diseases including virus infection, coronary artery disease, obesity and cancer. Here, we characterise ILRUN as a highly phylogenetically conserved protein containing UBA-like and a NBR1-like domains that are both essential for inhibition of type-I interferon and tumor necrosis factor alpha) transcription in human cells. We also solved the crystal structure of the NBR1-like domain, providing insights into its potential role in ILRUN function. This study provides critical information for future investigations into the role of ILRUN in health and disease.
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We used dynamic light scattering to detect aggregation of HIV-1 virions by antibodies (IgG) to the viral envelope glycoprotein (Env). Virions of different strains were inactivated by 2,2'-dithiodipyridine (AT-2), a procedure that abrogates infectivity but preserves the native antigenic structure of Env. Neutralizing antibodies directed to a V3-base- and glycan-dependent epitope on gp120 and to the apex of the Env trimer, as well as nonneutralizing antibodies to the epitope cluster I on the gp41-ectodomain, aggregated virions, but in markedly narrow concentration ranges. In contrast, the neutralizing antibody 2G12, which is specific for a composite glycan-dependent epitope on gp120 and functionally monovalent because of its unusual domain-swap structure, was nonaggregating. These results have potentially complex implications for vaccine development.
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Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Virión/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Anticuerpos Neutralizantes/inmunología , Dispersión Dinámica de Luz , Glicoproteínas , Inmunoglobulina G/inmunologíaRESUMEN
UNLABELLED: When HIV-1 vaccine candidates that include soluble envelope glycoproteins (Env) are tested in humans and other species, the resulting antibody responses to Env are sifted for correlates of protection or risk. One frequently used assay measures the reduction in antibody binding to Env antigens by an added chaotrope (such as thiocyanate). Based on that assay, an avidity index was devised for assessing the affinity maturation of antibodies of unknown concentration in polyclonal sera. Since a high avidity index was linked to protection in animal models of HIV-1 infection, it has become a criterion for evaluating antibody responses to vaccine candidates. But what does the assay measure and what does an avidity index mean? Here, we have used a panel of monoclonal antibodies to well-defined epitopes on Env (gp120, gp41, and SOSIP.664 trimers) to explore how the chaotrope acts. We conclude that the chaotrope sensitivity of antibody binding to Env depends on several properties of the epitopes (continuity versus tertiary- and quaternary-structural dependence) and that the avidity index has no simple relationship to antibody affinity for functional Env spikes on virions. We show that the binding of broadly neutralizing antibodies against quaternary-structural epitopes is particularly sensitive to chaotrope treatment, whereas antibody binding to epitopes in variable loops and to nonneutralization epitopes in gp41 is generally resistant. As a result of such biases, the avidity index may at best be a mere surrogate for undefined antibody or other immune responses that correlate weakly with protection. IMPORTANCE: An effective HIV-1 vaccine is an important goal. Such a vaccine will probably need to induce antibodies that neutralize typically transmitted variants of HIV-1, preventing them from infecting target cells. Vaccine candidates have so far failed to induce such antibody responses, although some do protect weakly against infection in animals and, possibly, humans. In the search for responses associated with protection, an avidity assay based on chemical disruption is often used to measure the strength of antibody binding. We have analyzed this assay mechanistically and found that the epitope specificity of an antibody has a greater influence on the outcome than does its affinity. As a result, the avidity assay is biased toward the detection of some antibody specificities while disfavoring others. We conclude that the assay may yield merely indirect correlations with weak protection, specifically when Env vaccination has failed to induce broad neutralizing responses.
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Afinidad de Anticuerpos , Anticuerpos Anti-VIH/inmunología , Anticuerpos Anti-VIH/metabolismo , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Vacunas contra el SIDA/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Biomarcadores , Epítopos/inmunología , Epítopos/metabolismo , Humanos , Inmunoensayo/métodos , Unión ProteicaRESUMEN
Bovine colostrum (first milk) contains very high concentrations of IgG, and on average 1 kg (500 g/liter) of IgG can be harvested from each immunized cow immediately after calving. We used a modified vaccination strategy together with established production systems from the dairy food industry for the large-scale manufacture of broadly neutralizing HIV-1 IgG. This approach provides a low-cost mucosal HIV preventive agent potentially suitable for a topical microbicide. Four cows were vaccinated pre- and/or postconception with recombinant HIV-1 gp140 envelope (Env) oligomers of clade B or A, B, and C. Colostrum and purified colostrum IgG were assessed for cross-clade binding and neutralization against a panel of 27 Env-pseudotyped reporter viruses. Vaccination elicited high anti-gp140 IgG titers in serum and colostrum with reciprocal endpoint titers of up to 1 × 10(5). While nonimmune colostrum showed some intrinsic neutralizing activity, colostrum from 2 cows receiving a longer-duration vaccination regimen demonstrated broad HIV-1-neutralizing activity. Colostrum-purified polyclonal IgG retained gp140 reactivity and neutralization activity and blocked the binding of the b12 monoclonal antibody to gp140, showing specificity for the CD4 binding site. Colostrum-derived anti-HIV antibodies offer a cost-effective option for preparing the substantial quantities of broadly neutralizing antibodies that would be needed in a low-cost topical combination HIV-1 microbicide.
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Anticuerpos Neutralizantes/inmunología , Calostro/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA , Animales , Anticuerpos Monoclonales/inmunología , Bovinos , Productos del Gen env/inmunología , Anticuerpos Anti-VIH/sangre , Infecciones por VIH/inmunología , Infecciones por VIH/prevención & control , Infecciones por VIH/virología , Inmunoglobulina G/inmunología , Pruebas de Neutralización , VacunaciónRESUMEN
Small non-coding micro-RNAs (miRNA) are important post-transcriptional regulators of mammalian gene expression that can be used to direct the knockdown of expression from targeted genes. We examined whether DNA vaccine vectors co-expressing miRNA with HIV-1 envelope (Env) antigens could influence the magnitude or quality of the immune responses to Env in mice. Human miR-155 and flanking regions from the non-protein encoding gene mirhg155 were introduced into an artificial intron within an expression vector for HIV-1 Env gp140. Using the miR-155-expressing intron as a scaffold, we developed novel vectors for miRNA-mediated targeting of the cellular antiviral proteins PKR and PERK, which significantly down-modulated target gene expression and led to increased Env expression in vitro. Finally, vaccinating BALB/c mice with a DNA vaccine vector delivering miRNA targeting PERK, but not PKR, was able to augment the generation of Env-specific T-cell immunity. This study provides proof-of-concept evidence that miRNA effectors incorporated into vaccine constructs can positively influence vaccine immunogenicity. Further testing of vaccine-encoded miRNA will determine if such strategies can enhance protective efficacy from vaccines against HIV-1 for eventual human use.
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VIH-1/inmunología , Inmunidad Celular/inmunología , MicroARNs/metabolismo , Vacunas de ADN/inmunología , eIF-2 Quinasa/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Vacunas contra el SIDA/inmunología , Animales , Antivirales/metabolismo , Activación Enzimática , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genes Dominantes/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , MicroARNs/genética , Proteínas de Unión al ARN/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The mechanism behind the positive action of introns upon transcription and the biological significance of this positive feedback remains unclear. Functional ablation of splice sites within an HIV-derived env cDNA significantly reduced transcription that was rescued by a U1 snRNA modified to bind to the mutated splice donor (SD). Using this model we further characterized both the U1 and pre-mRNA structural requirements for transcriptional enhancement. U1 snRNA rescued as a mature Sm-type snRNP with an intact stem loop II. Position and sequence context for U1-binding is crucial because a promoter proximal intron placed upstream of the mutated SD failed to rescue transcription. Furthermore, U1-rescue was independent of promoter and exon sequence and is partially replaced by the transcription elongation activator Tat, pointing to an intron-localized block in transcriptional elongation. Thus, transcriptional coupling of U1 snRNA binding to the SD may licence the polymerase for transcription through the intron.
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Intrones , Sitios de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Transcripción Genética , Secuencia de Bases , Sitios de Unión , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Empalme del ARN , Ribonucleoproteína Nuclear Pequeña U1/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
RNA interference (RNAi) is now widely used for gene silencing in mammalian cells. The mechanism uses the RNA-induced silencing complex, in which Dicer, Ago2, and the human immunodeficiency virus type 1 (HIV-1) TAR RNA binding protein (TRBP) are the main components. TRBP is a protein that increases HIV-1 expression and replication by inhibition of the interferon-induced protein kinase PKR and by increasing translation of viral mRNA. After HIV infection, TRBP could restrict the viral RNA through its activity in RNAi or could contribute more to the enhancement of viral replication. To determine which function will be predominant in the virological context, we analyzed whether the inhibition of its expression could enhance or decrease HIV replication. We have generated small interfering RNAs (siRNAs) against TRBP and found that they decrease HIV-1 long terminal repeat (LTR) basal expression 2-fold, and the LTR Tat transactivated level up to 10-fold. In the context of HIV replication, siRNAs against TRBP decrease the expression of viral genes and inhibit viral production up to fivefold. The moderate increase in PKR expression and activation indicates that it contributes partially to viral gene inhibition. The moderate decrease in micro-RNA (miRNA) biogenesis by TRBP siRNAs suggests that in the context of HIV replication, TRBP functions other than RNAi are predominant. In addition, siRNAs against Dicer decrease viral production twofold and impede miRNA biogenesis. These results suggest that, in the context of HIV replication, TRBP contributes mainly to the enhancement of virus production and that Dicer does not mediate HIV restriction by RNAi.