Biology subjects

Sourimant, J.

Publications and source records attributed to Sourimant, J..

9 recordsLinked to original sources

bioRxiv2026

A single methylation site regulates HRSV nucleocapsid architecture and replication

The nucleoprotein N of the human respiratory syncytial virus (HRSV) encases the viral genome, forming a flexible N-RNA nucleocapsid helix that serves as template for the viral polymerase L. Recent structural analysis revealed a non-canonical helical nucleocapsid arrangement that modulates RNA accessibility, yet its impact on polymerase function remains unknown. Here, we identified symmetric dimethylation of residue R27 of N as a critical modulator of nucleocapsid architecture and viral replication. We also showed that the methylase PRMT5 interacts with N and likely catalyses R27 methylation. Molecular dynamics simulations of RNA-free N dimers indicate that R27 methylation enhances opening and closing of the RNA-binding cavity, whereas a methylation-mimicking R27M substitution favours a closed state. Cryo-electron microscopy reveals a canonical, markedly straight R27M helix with increased rise and pitch. These findings demonstrate that post-translational modifications fine-tune interactions between N protomers, shaping nucleocapsid assembly, structure and dynamics, and thereby controlling HRSV replication.

microbiology↗

bioRxiv2025

Pre-assembly of biomolecular condensate seeds drives RSV replication

During infection many RNA viruses, including respiratory syncytial virus (RSV), form specialized biomolecular condensates, inclusion bodies (IBs), where viral transcription and replication occur1-4. Paradoxically, high protein concentrations are typically required for condensate nucleation5, yet attaining sufficient protein levels in infection is thought to require IBs for viral transcription and replication. To uncover how viruses solve this paradox to establish IBs, we visualized early infection of RSV in real-time with single genomic viral ribonucleoprotein (vRNP) resolution. Our results reveal that IBs are nucleated from infecting vRNPs rather than de novo in the cytoplasm. IB nucleation further requires in-virion pre-assembly of viral protein-protein interaction networks on vRNPs to form pre-replication centers (PRCs). PRCs are potent condensate nucleation seeds due to their resistance to disassembly and efficient recruitment of newly-synthesized viral proteins. The high protein affinity of PRCs also results in increased polymerase complex association, allowing efficient viral transcription even in the absence of IBs. Together, these activities create a feed-forward loop that drives rapid IB formation. Intriguingly, PRC assembly depends on in-virion viral protein levels and is highly heterogeneous among virions, explaining cell-to-cell heterogeneity in infection progression, and identifying heterogeneous virions as the origin of infection heterogeneity. Together, our results show that in-virion pre-assembly of PRCs kick-starts viral condensate nucleation upon host-cell entry, and explains cell-to-cell heterogeneity in RSV infection.

microbiology↗

bioRxiv2025

Can Plitidepsin Be Used as an Antiviral Against RSV?

Human respiratory syncytial virus (HRSV) is a main cause of acute lower respiratory tract infections in infants, the elderly and the immunocompromised patients. Although vaccines have recently been approved for the elderly and for pregnant women, there is no curative treatment for HRSV. HRSV replicates in the cytoplasm of infected cells, and transcription and replication of the viral genome depend on the viral RNA polymerase complex, which recruits cellular factors for RNA synthesis. Among them, the eucaryotic translation elongation factor 1A (eEF1A) was previously shown to be critical for HRSV replication. eEF1A activity can be inhibited by plitidepsin (Aplidin), a cyclopeptide extracted from ascidian Aplidium albicans, which was shown highly potent against SARS-CoV-2, with a 50% inhibitory concentration (IC90) of 0.70 to 1.62 nM depending on the cell line. Here, we investigated whether plitidepsin could also inhibit HRSV replication. We found that plitidepsin inhibited HRSV replication with an IC50 of {approx}3 nM in cell cultures. However, further investigation revealed that plitidepsin has pleiotropic effects, affecting the translation of both cellular and viral proteins in a similar manner. Overall, our results show that plitidepsin blocks cellular translation and indicate that plitidepsin induces a proteasome-mediated degradation of eEF1A, also showing the dependance of HRSV replication to cellular factors such as eEF1A. These results thus highlight an original mechanism of action of plitidepsin on eEF1A, which render the use of this compound for antiviral therapy very risky.

microbiology↗

bioRxiv2022

4-Fluorouridine mitigates lethal infection with pandemic human and highly pathogenic avian influenza viruses

Influenza outbreaks are associated with substantial morbidity, mortality and economic burden. Next generation antivirals are needed to treat seasonal infections and prepare against zoonotic spillover of avian influenza viruses with pandemic potential. Having previously identified oral efficacy of the nucleoside analog 4-Fluorouridine (4-FlU, EIDD-2749) against SARS-CoV-2 and respiratory syncytial virus, we explored activity of the compound against seasonal and highly pathogenic influenza (HPAI) viruses in cell culture, human airway epithelium organoids, and/or two animal models, ferrets and mice, that assess IAV transmission and lethal viral pneumonia, respectively. 4-FlU inhibited a panel of relevant influenza A and B viruses with nanomolar potency in organoids. In vitro polymerase assays revealed immediate chain termination of IAV polymerase after 4-FlU incorporation, in contrast to delayed chain termination of SARS-CoV-2 and RSV polymerase. Once-daily oral treatment of ferrets with 2 mg/kg 4-FlU initiated 12 hours after infection rapidly stopped virus shedding and prevented direct-contact transmission to untreated sentinels. Treatment of mice infected with a lethal inoculum of pandemic A/CA/07/2009 (H1N1)pdm09 (Ca09) with 2 mg/kg 4-FlU alleviated pneumonia. Three doses mediated complete survival when treatment was initiated up to 60 hours after infection, indicating an unusually broad window for effective intervention. Therapeutic oral 4-FlU ensured survival of animals infected with HPAI A/VN/12/2003 (H5N1) and of immunocompromised mice infected with pandemic Ca09. Recoverees were fully protected against homologous reinfection. This study defines the mechanistic foundation for high sensitivity of influenza viruses to 4-FlU and supports 4-FlU as developmental candidate for the treatment of seasonal and pandemic influenza. Author SummaryNext-generation antiviral therapeutics are needed to better mitigate seasonal influenza and prepare against zoonotic virus spillover from animal reservoirs. At greatest risk are the immunocompromised and patients infected with highly pathogenic influenza viruses. In this study, we have demonstrated efficacy of a broad-spectrum nucleoside analog, 4-fluorouridine, against a representative panel of influenza viruses in cell culture, human organoids, and two animal models, ferrets and mice. Acting as an immediate chain terminator of the influenza virus polymerase, once-daily oral treatment protected against lethal infection with seasonal and highly pathogenic avian influenza viruses, prevented direct-contact transmission to untreated sentinels, and mitigated lethal infection of immunocompromised hosts. These results support the developmental potential of 4-fluorouridine for treatment of vulnerable patient groups and mitigation of pandemic influenza, providing a much-needed additional therapeutic option for improved disease management.

microbiology↗

bioRxiv2022

SARS-CoV-2 variant of concern type and biological sex affect efficacy of molnupiravir in dwarf hamster model of severe COVID-19

Summary ParagraphSARS-CoV-2 variants of concern (VOC) have triggered distinct infection waves in the coronavirus disease 2019 (COVID-19) pandemic, culminating in currently all-time high incidence rates of VOC omicron. Orally available direct-acting antivirals such as molnupiravir promise to improve disease management and limit SARS-CoV-2 spread. However, molnupiravir efficacy against VOC delta was questioned based on clinical trial results and its potency against omicron is unknown. This study evaluates molnupiravir against a panel of relevant VOC in three efficacy models: primary human airway epithelium organoids, the ferret model of upper respiratory disease, and a lethal Roborovski dwarf hamster efficacy model of severe COVID-19-like acute lung injury. All VOC were equally efficiently inhibited by molnupiravir in cultured cells and organoids. Treatment consistently reduced upper respiratory VOC shedding in ferrets and prevented viral transmission. Pathogenicity in the dwarf hamsters was VOC-dependent and highest for gamma, omicron, and delta with fulminant lung histopathology. Oral molnupiravir started 12 hours after infection resulted in complete survival of treated dwarf hamsters independent of challenge VOC. However, reduction in lung virus differed VOC-dependently, ranging from one (delta) to four (gamma) orders of magnitude compared to vehicle-treated animals. Dwarf hamsters infected with VOC omicron showed significant individual variation in response to treatment. Virus load reduction was significant in treated males, but not females. The dwarf hamster model recapitulates mixed efficacy of molnupiravir seen in human trials and alerts that therapeutic benefit of approved antivirals must be continuously reassessed in vivo as new VOC emerge.

microbiology↗

bioRxiv2021

A bifluorescent-based assay for the identification of neutralizing antibodies against SARS-CoV-2 variants of concern in vitro and in vivo

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the United States (US) Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2 neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS- CoV-2 variants of concern (VoC). Here we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and a rSARS-CoV-2 expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta, {beta}) VoC, in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent- expressing rSARS-CoV-2 and the parental wild-type (WT) rSARS-CoV-2 WA-1 had similar viral fitness in vitro, as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS- CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection.

microbiology↗

bioRxiv2021

4'-Fluorouridine is a broad-spectrum orally efficacious antiviral blocking respiratory syncytial virus and SARS-CoV-2 replication

The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and the elderly. We describe 4-fluorouridine (4-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and SARS-CoV-2 with high selectivity index in cells and well-differentiated human airway epithelia. Polymerase inhibition in in vitro RdRP assays established for RSV and SARS-CoV-2 revealed transcriptional pauses at positions i or i+3/4 post-incorporation. Once-daily oral treatment was highly efficacious at 5 mg/kg in RSV-infected mice or 20 mg/kg in ferrets infected with SARS-CoV-2 WA1/2020 or variant-of-concern (VoC) isolate CA/2020, initiated 24 or 12 hours after infection, respectively. These properties define 4-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2 and related RNA virus infections. One-Sentence Summary4-Fluorouridine is an orally available ribonucleoside analog that efficiently treats RSV and SARS-CoV-2 infections in vivo.

microbiology↗

bioRxiv2021

SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern induce lethal disease in K18-hACE2 transgenic mice despite convalescent plasma therapy

SARS-CoV-2 variants of concern (VoCs) are impacting responses to the COVID-19 pandemic. Here we present a comparison of the SARS-CoV-2 USA-WA1/2020 (WA-1) strain with B.1.1.7 and B.1.351 VoCs and identify significant differences in viral propagation in vitro and pathogenicity in vivo using K18-hACE2 transgenic mice. Passive immunization with plasma from an early pandemic SARS-CoV-2 patient resulted in significant differences in the outcome of VoC-infected mice. WA-1-infected mice were protected by plasma, B.1.1.7-infected mice were partially protected, and B.1.351-infected mice were not protected. Serological correlates of disease were different between VoC-infected mice, with B.1.351 triggering significantly altered cytokine profiles than other strains. In this study, we defined infectivity and immune responses triggered by VoCs and observed that early 2020 SARS-CoV-2 human immune plasma was insufficient to protect against challenge with B.1.1.7 and B.1.351 in the mouse model.

microbiology↗

bioRxiv2020

Therapeutic Targeting of Measles Virus Polymerase with ERDRP-0519Suppresses All RNA Synthesis Activity

Morbilliviruses, such as measles virus (MeV) and canine distemper virus (CDV), are highly infectious members of the paramyxovirus family. MeV is responsible for major morbidity and mortality in non-vaccinated populations. ERDRP-0519, a pan-morbillivirus small molecule inhibitor for the treatment of measles, targets the morbillivirus RNA-dependent RNA-polymerase (RdRP) complex and displayed unparalleled oral efficacy against lethal infection of ferrets with CDV, an established surrogate model for human measles. Resistance profiling identified the L subunit of the RdRP, which harbors all enzymatic activity of the polymerase complex, as the molecular target of inhibition. Here, we examined binding characteristics, physical docking site, and the molecular mechanism of action of ERDRP-0519 through label-free biolayer interferometry, photoaffinity cross-linking, and in vitro RdRP assays using purified MeV RdRP complexes and synthetic templates. Results demonstrate that unlike all other mononegavirus small molecule inhibitors identified to date, ERDRP-0519 inhibits all phosphodiester bond formation in both de novo initiation of RNA synthesis at the promoter and RNA elongation by a committed polymerase complex. Photocrosslinking and resistance profiling-informed ligand docking revealed that this unprecedented mechanism of action of ERDRP-0519 is due to simultaneous engagement of the L protein polyribonucleotidyl transferase (PRNTase)-like domain and the flexible intrusion loop by the compound, pharmacologically locking the polymerase in pre-initiation conformation. This study informs selection of ERDRP-0519 as clinical candidate for measles therapy and identifies a previously unrecognized druggable site in mononegavirus L polymerase proteins that can silence all synthesis of viral RNA. ImportanceThe mononegavirus order contains major established and recently emerged human pathogens. Despite the threat to human health, antiviral therapeutics directed against this order remain understudied. The mononegavirus polymerase complex represents a promising drug target due to its central importance for both virus replication and viral mitigation of the innate host antiviral response. In this study, we have mechanistically characterized a clinical candidate small-molecule MeV polymerase inhibitor. The compound blocked all phosphodiester bond formation activity, a unique mechanism of action unlike all other known mononegavirus polymerase inhibitors. Photocrosslinking-based target site mapping demonstrated that this class-defining prototype inhibitor stabilizes a pre-initiation conformation of the viral polymerase complex that sterically cannot accommodate template RNA. Function-equivalent druggable sites exist in all mononegavirus polymerases. In addition to its direct anti-MeV impact, the insight gained in this study can therefore serve as a blueprint for indication spectrum expansion through structure-informed scaffold engineering or targeted drug discovery.

microbiology↗