A Simple and Fast Method to Sequence the Full-Length Spike Gene for SARS-CoV-2 Variant Identification from Patient Samples

Since the beginning of the pandemic, a race has been underway to detect SARS-CoV-2 virus infection (PCR screening, serological diagnostic kits), treat patients (drug repurposing, standard care) and develop a vaccine. After almost a year of active circulation worldwide, SARS-CoV-2 variants have appeared in different countries. Those variants include mutations in multiple regions of the genome, particularly in the spike gene. Because this surface protein is a key player in both the spread of the virus and the efficacy of vaccine strategies, the challenge is to efficiently monitor the appearance of spike mutations in the population. The present work describes a procedure based on the widely available Sanger technology to produce a full-length sequence of the spike gene from patient-derived samples

Absence of Resistance Mutations in the Integrase Coding Region among ART-Experienced Patients in the Republic of the Congo

Background: HIV infects around one hundred thousand patients in the Republic of the Congo. Approximately 25% of them receive an antiretroviral treatment; current first-line regimens include two NRTIs and one NNRTI, reverse transcriptase inhibitors. Recently, protease inhibitors (PIs) were also introduced as second-line therapy upon clinical signs of treatment failure. Due to the limited number of molecular characterizations and amount of drug resistance data available in the Republic of the Congo, this study aims to evaluate the prevalence of circulating resistance mutations within the pol region. Methods: HIV-positive, ART-experienced patients have been enrolled in four semi-urban localities in the Republic of the Congo. Plasma samples were collected, and viral RNA was extracted. The viral load for each patient was evaluated by RT-qPCR, following the general diagnostic procedures of the University Hospital of Bordeaux. Finally, drug resistance genotyping and phylogenetic analysis were conducted following Sanger sequencing of the pol region. Results: A high diversity of HIV-1 strains was observed with many recombinant forms. Drug resistance mutations in RT and PR genes were determined and correlated to HAART. Because integrase inhibitors are rarely included in treatments in the Republic of the Congo, the prevalence of integrase drug resistance mutations before treatment was also determined. Interestingly, very few mutations were observed. Conclusions: We confirmed a high diversity of HIV-1 in the Republic of the Congo. Most patients presented an accumulation of mutations conferring resistance against NRTIs, NNRTIs and PIs. Nonetheless, the absence of integrase mutations associated with drug resistance suggests that the introduction of integrase inhibitors into therapy will be highly beneficial to patients in the Republic of the Congo

Potential antiviral effects of pantethine against SARS-CoV-2

Abstract SARS-CoV-2 interacts with cellular cholesterol during many stages of its replication cycle. Pantethine was reported to reduce total cholesterol levels and fatty acid synthesis and potentially alter different processes that might be involved in the SARS-CoV-2 replication cycle. Here, we explored the potential antiviral effects of pantethine in two in vitro experimental models of SARS-CoV-2 infection.Pantethine reduced the infection of cells by SARS-CoV-2 in both preinfection and postinfection treatment regimens. Accordingly, cellular expression of the viral spike and nucleocapsid proteins was substantially reduced, and we observed a significant reduction in viral copy numbers in the supernatant of cells treated with pantethine. In addition, pantethine inhibited the infection-induced increase in TMPRSS2 and HECT E3 ligase expression in infected cells as well as the increase in antiviral interferon-beta response and inflammatory gene expression in Calu-3a cells. Our results demonstrate that pantethine, which is well tolerated in humans, was very effective in controlling SARS-CoV-2 infection and might represent a new therapeutic drug that can be repurposed for the prevention or treatment of COVID-19 and long COVID syndrome

SARS-CoV-2 transmission via apical syncytia release from primary bronchial epithelia and infectivity restriction in children epithelia

The beta-coronavirus SARS-CoV-2 is at the origin of a persistent worldwide pandemic. SARS-CoV-2 infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract eventually causing acute severe respiratory syndrome with a high degree of mortality in the elderly. Here we use reconstituted primary bronchial epithelia from adult and children donors to follow the infection dynamic following infection with SARS-CoV-2. We show that in bronchial epithelia derived from adult donors, infections initiate in multi-ciliated cells. Then, infection rapidly spread within 24-48h throughout the whole epithelia. Within 3-4 days, large apical syncytia form between multi-ciliated cells and basal cells, which dissipate into the apical lumen. We show that these syncytia are a significant source of the released infectious dose. In stark contrast to these findings, bronchial epithelia reconstituted from children donors are intrinsically more resistant to virus infection and show active restriction of virus spread. This restriction is paired with accelerated release of IFN compared to adult donors. Taken together our findings reveal apical syncytia formation as an underappreciated source of infectious virus for either local dissemination or release into the environment. Furthermore, we provide direct evidence that children bronchial epithelia are more resistant to infection with SARS-CoV-2 providing experimental support for epidemiological observations that SARS-CoV-2 cases’ fatality is linked to age. Significance Statement Bronchial epithelia are the primary target for SARS-CoV-2 infections. Our work uses reconstituted bronchial epithelia from adults and children. We show that infection of adult epithelia with SARS-CoV-2 is rapid and results in the synchronized release of large clusters of infected cells and syncytia into the apical lumen contributing to the released infectious virus dose. Infection of children derived bronchial epithelia revealed an intrinsic resistance to infection and virus spread, probably as a result of a faster onset of interferon secretion. Thus, our data provide direct evidence for the epidemiological observation that children are less susceptible to SARS-CoV-2

Structure and Functional Analysis of the RNA- and Viral Phosphoprotein-Binding Domain of Respiratory Syncytial Virus M2-1 Protein

Respiratory syncytial virus (RSV) protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-158–177 core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-158–177, as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1

Etude structurale et fonctionnelle du facteur de transcription M2-1 du virus respiratoire syncytial

Le virus respiratoire syncytial (VRS) est le principal agent responsable de maladies respiratoires graves chez les jeunes enfants. Il n y a pas de vaccin pour l homme et le développement d antiviraux est nécessaire. Le génome du VRS est constitué d un ARN simple brin de polarité négative qui sert de matrice pour la transcription et la réplication des ANR viraux. L ARN polymérase virale est un complexe composé de protéines N, L, P, et M2-1 est capable de se lier à l ARN mais aussi d interagir avec la phosphoprotéine P. Ces propriétés de liaison sont liées à la région globulaire de M2-1 allant de la Sérine 58 à l Arginine 177. Nous avons déterminé la structure de ce domaine par résonance magnétique nucléaire (RMN). Le recouvrement partiel des surfaces d interaction pour l ARN et P sur M2-158-177 confirme les résultats obtenus préalablement concernant une compétition entre ces deux partenaires. Nous avons identifié huit résidus localisés sur ces surfaces critiques pour l activité transcriptionnelle du complexe polymérase. Des mutations simples suffisent à perturber spécifiquement soit l interaction avec P soit avec l ARN sur M2-1. Le recrutement de M2-1 dans le corps d inclusion cytoplasmiques, correspondant à des sites de synthèse de l ARN viral, est diminué par des mutations affectant uniquement l interaction avec P. Ces résultats révèlent que les interactions entre M2-1 et l ARN ou P peuvent être découplées bien qu elles soient essentielles pour la fonction d activateur de la transcription de M2-1.Respiratory Syncytial Virus (RSV) is the main cause of pneumonia and bronchiolitis in young children. There is no vaccine nor antiviral for humans. This virus encodes its own NA-dependent RNA polymerase (RdRp) to transcribe and replicate viral genes.The RdRp is composed of the nucleoprotein N, the phosphoprotein P, the large protein L and the transcription factor M2-1. Our aim is to characterize the structure and the function of the RdRp proteins to develop antiviral strategies. The M2-1 protein functions as an essential transcriptional cofactor of the RdRp complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177.Here we report the NMR structure of the RSV M2-158-177 core domain. The partial overlap of RNA and P interaction surfaces on M2-158-177 rationalizes the previously observed competitive behavior of RNA versus P. we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single bodies, which are regarded as sites of viral RNA synthesis, was impared by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the hononucleocapsid by interacting with P.These results reveal that RNA and P binding to M2- can be uncoupled and that both are critical for the transcriptional activation function of M2-1.VERSAILLES-BU Sciences et IUT (786462101) / SudocSudocFranceF

Etude fonctionnelle de la protéine M2-1 du virus respiratoire syncytial à l'aide d'un miniréplicon en cellules eucaryotes

Le virus respiratoire syncytial (VRS) est le principal agent responsable de maladies respiratoires graves (bronchiolites, pneumonies) chez l’Homme (nouveau-nés, personnes âgées ou immunodéprimées). En l’absence de vaccins efficaces contre ce virus, le développement rationnel de traitements antiviraux constitue un enjeu de taille. Appartenant à la famille des Paramyxoviridae, le VRS est un virus enveloppé dont le génome est constitué d’un ARN simple brin de polarité négative, codant pour 11 protéines. Le génome est répliqué et transcrit par le complexe ARN polymérase dépendante de l’ARN viral. Ce complexe est composé de la nucléoprotéine N, de la polymérase L, de la phosphoprotéine P et du facteur de transcription M2-1 qui assure la processivité de la polymérase au cours de la transcription virale. Le complexe polymérase, ne présentant pas d’équivalent dans la cellule, constitue une cible privilégiée pour le développement d’inhibiteurs. Une meilleure compréhension des mécanismes d’interaction entre les protéines du complexe polymérase est donc indispensable afin d’identifier des cibles potentielles. Le groupe de John N. Barr (Leeds University, UK) a résolu la structure cristalline tridimensionnelle de la protéine M2-1, dans sa forme tétramérique avec une résolution de 2,5Å. Nous nous sommes associés à ce laboratoire afin étudier le rôle fonctionnel de certains résidus de la protéine M2-1. Le rôle critique de certains résidus a été abordé par mutagenèse dirigée et l’utilisation d’un miniréplicon accompagné d’un contrôle d’expression par western blotting des protéines mutées. Nous avons notamment montré l’importance de deux résidus Serine phosphorylables pour l’activité de M2-1 en tant que facteur de transcription

Mise au point d’un nouvel outil de génétique inverse pour le Virus Respiratoire Syncytial humain et perspectives.

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