Biology subjects

Clementi, M.

Publications and source records attributed to Clementi, M..

11 recordsLinked to original sources

bioRxiv2022

Discovering host protein interactions specific for SARS-CoV-2 RNA genome

SARS-CoV-2, a positive single-stranded RNA virus, interacts with host cell proteins throughout its life cycle. These interactions are necessary for the host to recognize and hinder the replication of SARS-CoV-2. For the virus, to translate, transcribe and replicate its genetic material. However, many details of these interactions are still missing. We focused on the proteins binding to the highly structured 5 and 3 end regions of SARS-CoV-2 RNA that were predicted by the catRAPID algorithm to attract numerous proteins, exploiting RNA-Protein Interaction Detection coupled with Mass Spectrometry (RaPID-MS) technology. The validated interactors, which agreed with our predictions, include pseudouridine synthase PUS7 that binds to both ends of the viral RNA. Nanopore direct-RNA sequencing confirmed that the RNA virus is heavily modified, and PUS7 consensus regions were found in both SARS-CoV-2 RNA end regions. Notably, a modified site was detected in the viral Transcription Regulatory Sequence - Leader (TRS-L) and can influence the viral RNA structure and interaction propensity. Overall, our data map host protein interactions within SARS-CoV-2 UTR regions, pinpointing to a potential role of pseudouridine synthases and post-transcriptional modifications in the viral life cycle. These findings contribute to understanding virus-host dynamics and may guide the development of targeted therapies.

molecular biology↗

bioRxiv2021

Nanopore ReCappable Sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested sub genomic RNAs used to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

genomics↗

bioRxiv2021

SARS-CoV-2 Spike Affinity and Dynamics Exclude the Strict Requirement of an Intermediate Host

SARS-CoV-2 proximal origin is still unclear, limiting the possibility of foreseeing other spillover events with pandemic potential. Here we propose an evolutionary model based on the thorough dissection of SARS-CoV-2 and RaTG13 - the closest bat relative - spike dynamics, kinetics and binding to ACE2. Our results indicate that both spikes share nearly identical, high affinities for Rhinolophus affinis bat and human ACE2, pointing out to negligible species barriers directly related to receptor binding. Also, SARS-CoV-2 spike shows a higher degree of dynamics and kinetics optimization that favors ACE2 engagement. Therefore, we devise an affinity-independent evolutionary process that likely took place in R. affinis bats and limits the eventual involvement of other animal species in initiating the pandemic to the role of vector.

microbiology↗

bioRxiv2021

Differential plasmacytoid dendritic cell phenotype and type I Interferon response in asymptomatic and severe COVID-19 infection

SARS-CoV-2 fine-tunes the interferon (IFN)-induced antiviral responses, which play a key role in preventing coronavirus disease 2019 (COVID-19) progression. Indeed, critically ill patients show an impaired type I IFN response accompanied by elevated inflammatory cytokine and chemokine levels, responsible for cell and tissue damage and associated multi-organ failure. Here, the early interaction between SARS-CoV-2 and immune cells was investigated by interrogating an in vitro human peripheral blood mononuclear cell (PBMC)-based experimental model. We found that, even in absence of a productive viral replication, the virus mediates a vigorous TLR7/8-dependent production of both type I and III IFNs and inflammatory cytokines and chemokines, known to contribute to the cytokine storm observed in COVID-19. Interestingly, we observed how virus-induced type I IFN secreted by PBMC enhances anti-viral response in infected lung epithelial cells, thus, inhibiting viral replication. This type I IFN was released by plasmacytoid dendritic cells (pDC) via an ACE-2-indipendent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PD-L1 marker, a feature of type I IFN producing cells. Coherently to what observed in vitro, asymptomatic SARS-CoV-2 infected subjects displayed a similar pDC phenotype associated to a very high serum type I IFN level and induction of anti-viral IFN-stimulated genes in PBMC. Conversely, hospitalized patients with severe COVID-19 display very low frequency of circulating pDC with an inflammatory phenotype and high levels of chemokines and pro-inflammatory cytokines in serum. This study further shed light on the early events resulting from the interaction between SARS-CoV-2 and immune cells occurring in vitro and confirmed ex vivo. These observations can improve our understanding on the contribution of pDC/type I IFN axis in the regulation of the anti-viral state in asymptomatic and severe COVID-19 patients. Author summarySARS-CoV-2 pandemic has resulted in millions of infections and deaths worldwide, yet the role of host innate immune responses in COVID-19 pathogenesis remains only partially characterized. Innate immunity represents the first line of host defense against viruses. Upon viral recognition, the secretion of type I and III interferons (IFN) establishes the cellular state of viral resistance, and contributes to induce the specific adaptive immune responses. Moving from in vitro evidences on the protective role played by plasmacytoid dendritic cells (pDC)-released type I IFN in the early phase of SARS-CoV-2 infection, here we characterized ex vivo the pDC phenotype and the balance between anti-viral and pro-inflammatory cytokines of COVID-19 patients stratified according to disease severity. Our study confirms in COVID-19 the crucial and protective role of pDC/type I IFN axis, whose deeper understanding may contribute to the development of novel pharmacological strategies and/or host-directed therapies aimed at boosting pDC response since the early phases of SARS-CoV-2 infection.

immunology↗

bioRxiv2021

Severity of SARS-CoV-2 infection as a function of the interferon landscape across the respiratory tract of COVID-19 patients.

The COVID-19 outbreak driven by SARS-CoV-2 has caused more than 2.5 million deaths globally, with the most severe cases characterized by over-exuberant production of immune-mediators, the nature of which is not fully understood. Interferons of the type I (IFN-I) or type III (IFN-III) families are potent antivirals, but their role in COVID-19 remains debated. Our analysis of gene and protein expression along the respiratory tract shows that IFNs, especially IFN-III, are over-represented in the lower airways of patients with severe COVID-19, while high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity; also, IFN expression varies with abundance of the cell types that produce them. Our data point to a dynamic process of inter- and intra-family production of IFNs in COVID-19, and suggest that IFNs play opposing roles at distinct anatomical sites.

immunology↗

bioRxiv2020

A human stem cell-derived neurosensory-epithelial circuitry on-a-chip to model herpes simplex virus reactivation

Both emerging viruses and well-known viral pathogens endowed with neurotropism can either impair directly the neuronal functions or induce physio-pathological changes by diffusing from the periphery through neurosensory-epithelial connections. However, the current lack of an in vitro system modeling the connectivity between human neurons and peripheral tissues excludes the analysis of viral latency and reactivation and the assessment of natural/artificial induced anti-viral immunity. In this study, we developed the first stable topographic neurosensory-epithelial connection on-a-chip using human stem cell derived dorsal root ganglia (DRG) sensory neurons. Bulk and single cell transcriptomics showed that different combinations of key receptors for Herpes Simplex Virus 1 (HSV-1) are expressed by each sensory neuronal cell type. This neuronal-epithelial circuitry enabled a detailed analysis of the HSV infectivity faithfully modeling its dynamics and cell type specificity. The reconstitution of an organized connectivity between human sensory neurons and keratinocytes into microfluidic chips provides for the first time a powerful in vitro platform to model viral latency and reactivation of human viral pathogens.

microbiology↗

bioRxiv2020

Vinegar and Its Active Component Acetic Acid Inhibit SARS-CoV-2 Infection In Vitro and Ex Vivo

Effective and economical measures are needed to either prevent or inhibit the replication of SARS-CoV-2, the causative agent of COVID-19, in the upper respiratory tract. As fumigation of vinegar at low concentration (0.34%) ameliorated the symptoms of mild SARS-CoV-2 infection, we tested in vitro the potential antiviral activity of vinegar and of its active component, acetic acid. We here demonstrate that both vinegar and acetic acid indeed strongly inactivate SARS-CoV-2 infectivity in Vero cells. Furthermore, vinegar treatment caused a 90% inhibition of the infectious titer when directly applied to a nasopharyngeal swab transfer medium of a COVID-19 patient. These effects were potentiated if conduced at a temperature of 45 {degrees}C vs. 37 {degrees}C, a condition that is transiently generated in the upper respiratory tract during fumigation. Our findings are consistent and extend the results of studies performed in the early and mid-20th century on the disinfectant capacity of organic acids and can provide an affordable home-made aid to prevent or contain SARS-CoV-2 infection of the upper respiratory tract.

microbiology↗

bioRxiv2020

Heparin inhibits cellular invasion by SARS-CoV-2: structural dependence of the interaction of the surface protein (spike) S1 receptor binding domain with heparin.

The dependence of the host on the interaction of hundreds of extracellular proteins with the cell surface glycosaminoglycan heparan sulphate (HS) for the regulation of homeostasis is exploited by many microbial pathogens as a means of adherence and invasion. The closely related polysaccharide heparin, the widely used anticoagulant drug, which is structurally similar to HS and is a common experimental proxy, can be expected to mimic the properties of HS. Heparin prevents infection by a range of viruses if added exogenously, including S-associated coronavirus strain HSR1. Heparin prevents infection by a range of viruses if added exogenously, including S-associated coronavirus strain HSR1. Here, we show that the addition of heparin to Vero cells between 6.25 - 200 g.ml-1, which spans the concentration of heparin in therapeutic use, and inhibits invasion by SARS-CoV-2 at between 44 and 80%. We also demonstrate that heparin binds to the Spike (S1) protein receptor binding domain and induces a conformational change, illustrated by surface plasmon resonance and circular dichroism spectroscopy studies. The structural features of heparin on which this interaction depends were investigated using a library of heparin derivatives and size-defined fragments. Binding is more strongly dependent on the presence of 2-O or 6-O sulphation, and the consequent conformational consequences in the heparin structure, than on N-sulphation. A hexasaccharide is required for conformational changes to be induced in the secondary structure that are comparable to those that arise from heparin binding. Enoxaparin, a low molecular weight clinical anticoagulant, also binds the S1 RBD protein and induces conformational change. These findings have implications for the rapid development of a first-line therapeutic by repurposing heparin as well as for next-generation, tailor-made, GAG-based antiviral agents against SARS-CoV-2 and other members of the Coronaviridae.

biochemistry↗

bioRxiv2020

Combined prophylactic and therapeutic use maximizes hydroxychloroquine anti-SARS-CoV-2 effects in vitro

While the SARS-CoV-2 pandemic is hardly hitting the world, it is of extreme importance that significant in vitro observations guide the quick set up of clinical trials. In this study, we evidence that the anti-SARS-CoV2 activity of a clinically achievable hydroxychloroquine concentration is maximized only when administered before and after the infection of Vero E6 cells. This strongly suggests that only a combined prophylactic and therapeutic use of hydroxychloroquine may be effective in limiting viral replication in patients.

microbiology↗

bioRxiv2019

Shared Genetic Risk between Eating Disorder- and Substance-Use-Related Phenotypes: Evidence from Genome-Wide Association Studies

Eating disorders and substance use disorders frequently co-occur. Twin studies reveal shared genetic variance between liabilities to eating disorders and substance use, with the strongest associations between symptoms of bulimia nervosa (BN) and problem alcohol use (genetic correlation [rg], twin-based=0.23-0.53). We estimated the genetic correlation between eating disorder and substance use and disorder phenotypes using data from genome-wide association studies (GWAS). Four eating disorder phenotypes (anorexia nervosa [AN], AN with binge-eating, AN without binge-eating, and a BN factor score), and eight substance-use-related phenotypes (drinks per week, alcohol use disorder [AUD], smoking initiation, current smoking, cigarettes per day, nicotine dependence, cannabis initiation, and cannabis use disorder) from eight studies were included. Significant genetic correlations were adjusted for variants associated with major depressive disorder (MDD). Total sample sizes per phenotype ranged from ~2,400 to ~537,000 individuals. We used linkage disequilibrium score regression to calculate single nucleotide polymorphism-based genetic correlations between eating disorder and substance-use-related phenotypes. Significant positive genetic associations emerged between AUD and AN (rg=0.18; false discovery rate q=0.0006), cannabis initiation and AN (rg=0.23; q<0.0001), and cannabis initiation and AN with binge-eating (rg=0.27; q=0.0016). Conversely, significant negative genetic correlations were observed between three non-diagnostic smoking phenotypes (smoking initiation, current smoking, and cigarettes per day) and AN without binge-eating (rgs=-0.19 to -0.23; qs<0.04). The genetic correlation between AUD and AN was no longer significant after co-varying for MDD loci. The patterns of association between eating disorder- and substance-use-related phenotypes highlights the potentially complex and substance-specific relationships between these behaviors.

genetics↗

bioRxiv2016

Genome-Wide Association Study Reveals First Locus for Anorexia Nervosa and Metabolic Correlations

Anorexia nervosa (AN) is a serious eating disorder characterized by restriction of energy intake relative to requirements, resulting in abnormally low body weight. It has a lifetime prevalence of approximately 1%, disproportionately affects females1,2, and has no well replicated evidence of effective pharmacological or psychological treatments despite high morbidity and mortality2. Twin studies support a genetic basis for the observed aggregation of AN in families3, with heritability estimates of 48%-74%4. Although initial genome-wide association studies (GWASs) were underpowered5,6, evidence suggested that signals for AN would be detected with increased power5. We present a GWAS of 3,495 AN cases and 10,982 controls with one genome-wide significant locus (index variant rs4622308, p=4.3x10-9) in a region (chr12:56,372,585-56,482,185) which includes six genes. The SNP-chip heritability [Formula] of AN from these data is 0.20 (SE=0.02), suggesting that a substantial fraction of the twin-based heritability stems from common genetic variation. Using these GWAS results, we also find significant positive genetic correlations with schizophrenia, neuroticism, educational attainment, and HDL cholesterol, and significant negative genetic correlations with body mass, insulin, glucose, and lipid phenotypes. Our results support the reconceptualization of AN as a disorder with both psychiatric and metabolic components.

genomics↗