Biology subjects

Palor, M.

Publications and source records attributed to Palor, M..

8 recordsLinked to original sources

bioRxiv2026

Antiviral drug synergy and mutational signatures in different epithelial cell models of RSV and hPIV infection

Despite the huge global health burden presented by respiratory viruses, effective broad-spectrum antiviral therapeutic options remain limited. Here we evaluated the antiviral activity of four RNA-dependent RNA polymerase (RdRp) inhibitors, remdesivir, ribavirin, favipiravir, and molnupiravir, against respiratory syncytial virus (RSV) and human parainfluenza (hPIV) using monotherapy or dual-drug combinations with epithelial cell lines and primary human airway culture models. Remdesivir showed the greatest potency across both viruses, while ribavirin and favipiravir also demonstrated inhibition. Molnupiravir was active against RSVA but not hPIV3. Several dual-drug combinations, including remdesivir-favipiravir, remdesivir-molnupiravir and favipiravir-molnupiravir, produced marked synergy against RSVA, and more limited synergy for hPIV3. Antiviral efficacy was validated in primary airway epithelial cultures, where effective concentrations preserved epithelial integrity and attenuated viral disruption of ciliary function. Across both viruses, increasing antiviral exposure was associated with dose-dependent signature mutagenesis. Importantly, antivirals induced significantly higher RSVA mutation burden in the primary airway model. These findings highlight the therapeutic potential of RdRp inhibitor combinations for RSVA and hPIV3, provide mechanistic insight through antiviral-related mutational signatures and demonstrate advantages of the primary human airway culture model for development of effective multi-drug regimens and broad-spectrum antiviral preparedness. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/699296v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1eb00dforg.highwire.dtl.DTLVardef@186358forg.highwire.dtl.DTLVardef@265181org.highwire.dtl.DTLVardef@1b10d0e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Neutrophil myeloperoxidase as a functional biomarker for RSV severity: implications for in vitro therapeutic screening.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections in infants, yet effective therapeutics are lacking. The aim of this study is to develop an in vitro model that recapitulates key clinical outcomes in infants with RSV bronchiolitis to help accelerate the discovery of effective therapeutics. Neutrophil activation and influx into the airways are hallmarks of severe RSV infection, but these responses are difficult to quantify in clinical trials. Here we profile peripheral blood-derived neutrophils from infants with RSV admitted to the Paediatric Intensive Care Unit (PICU) and identify myeloperoxidase (MPO) as a key indicator of disease severity when compared to age matched controls. To mechanistically model this response, we established a paediatric airway epithelial air-liquid interface (ALI) system incorporating an endothelial layer and primary neutrophils to recapitulate the tissue microenvironment at the blood-airway barrier. Following RSV infection, neutrophil migration and activation were assessed using flow cytometry. The inclusion of an endothelial layer enhanced physiological relevance and more accurately replicated in vivo MPO responses. We then evaluated two antiviral candidates (remdesivir (RDV) and RSV604) to assess their ability to modulate neutrophil activation. While both compounds reduced viral load at 24 hours post-infection, only RSV604 attenuated MPO expression. These findings establish MPO as both a biomarker of RSV disease severity and a functional readout of therapeutic efficacy and demonstrate that targeting neutrophildriven inflammatory pathways may be critical for reducing pathology in infant RSV infection. Take home messageAntiviral drug discovery should include neutrophil MPO reduction as a readout of therapeutic efficacy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/682115v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@170058corg.highwire.dtl.DTLVardef@17c344corg.highwire.dtl.DTLVardef@14d79baorg.highwire.dtl.DTLVardef@172ac3c_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

The emergence of goblet inflammatory or ITGB6hi nasal progenitor cells determines age-associated SARS-CoV-2 pathogenesis

Children infected with SARS-CoV-2 rarely progress to respiratory failure, but the risk of mortality in infected people over 85 years of age remains high, despite vaccination and improving treatment options. Here, we take a comprehensive, multidisciplinary approach to investigate differences in the cellular landscape and function of paediatric (<11y), adult (30- 50y) and elderly (>70y) nasal epithelial cells experimentally infected with SARS-CoV-2. Our data reveal that nasal epithelial cell subtypes show different tropism to SARS-CoV-2, correlating with age, ACE2 and TMPRSS2 expression. Ciliated cells are a viral replication centre across all age groups, but a distinct goblet inflammatory subtype emerges in infected paediatric cultures, identifiable by high expression of interferon stimulated genes and truncated viral genomes. In contrast, infected elderly cultures show a proportional increase in ITGB6hi progenitors, which facilitate viral spread and are associated with dysfunctional epithelial repair pathways. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/524211v2_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@dab12aorg.highwire.dtl.DTLVardef@1a57334org.highwire.dtl.DTLVardef@12e7983org.highwire.dtl.DTLVardef@2bbe6e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Distantly related hepaciviruses share common entry factor, Claudin-1

Due to increased and broadened screening efforts, the last decade has seen a rapid expansion in the number of viral species classified into the Hepacivirus genus. Conserved genetic features of hepaciviruses suggest they have undergone specific adaptation and evolved to hijack similar host proteins for efficient propagation in the liver. Here, we developed pseudotyped viruses to elucidate the entry factors of GB virus-B (GBV-B), the first hepacivirus described in an animal, closely related to hepatitis C virus (HCV). GBV-B pseudotyped viruses (GBVBpp) were shown to be uniquely sensitive to the serum of tamarins infected with GBV-B, validating their usefulness as a surrogate for GBV-B entry studies. We screened GBVBpp infection of hepatoma cell lines CRISPR/Cas9-engineered to ablate expression of individual HCV receptors/entry factors and found that claudin-1 is essential for GBV-B infection, indicating GBV-B and HCV share a common entry factor. Our data suggest that claudin-1 facilitates HCV and GBV-B entry through distinct mechanisms since the former requires the first extracellular loop and the latter is reliant on the second extracellular loop. The implication of this sharing of claudin-1 as an entry factor between these two hepaciviruses on their tissue tropism and evolutionary relationships will be discussed. ImportanceHepatitis C virus (HCV) is a major public health burden; approximately 58 million individuals have chronic HCV infection which could lead to cirrhosis and liver cancer. To achieve the World Health Organisation target of eliminating hepatitis by 2030, new therapeutics and vaccines are needed. Understanding how HCV enters the cells will inform the design of new vaccines and treatments, targeting the first stage of HCV infection. However, the entry mechanism is complex and sparsely described. Studying the entry of related hepaciviruses will increase the knowledge of the molecular mechanisms of the first stages of HCV infection, such as the membrane fusion, and inform structure-guided HCV vaccine design; in this work we have identified a protein, Claudin-1, which facilitates the entry of HCV-related hepacivirus, but with a mechanism not described for HCV. Similar work on other hepaciviruses may unveil commonality of entry factors and possibly new mechanisms.

microbiology↗

bioRxiv2021

Spatial and temporal analysis of neutrophil trans-epithelial migration in response to RSV infection

The recruitment of neutrophils to the infected airway occurs early following respiratory syncytial virus (RSV) infection and high numbers of activated neutrophils in airway and blood is associated with the development of severe disease. Here, we investigated whether trans-epithelial migration across primary human airway epithelial cells (AECs) is sufficient and necessary for neutrophil activation during RSV infection. Using flow cytometry, we identified three populations of neutrophils in our in vitro model; those in suspension in basolateral and apical compartments and those that migrated and adhered to AECs. After 1h incubation, the number of adherent neutrophils was significantly greater following RSV infection compared to mock infected. We found that, when migration occurred, neutrophil expression of CD11b, CD62L, CD64, NE and MPO increased in all compartments. However, this did not occur when neutrophils were prevented from migrating. This suggests that the heightened neutrophil activation we detected in the basolateral compartment may be due to reverse migrating neutrophils, as has been suggested by clinical observations. Using live-cell fluorescent microscopy, we then profiled the early temporal and spatial movement and adherence of human neutrophils during migration. Our findings suggest three main phases of early neutrophil recruitment and behaviour in the airways during RSV infection, with neutrophil recruitment, activation and adherence to RSV infected AECs, with clustering, occurring within the first 20 minutes. This work and the model we developed could provide new insight into how neutrophil activation and a dysregulated neutrophil response to RSV mediates disease severity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/463016v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@16639c5org.highwire.dtl.DTLVardef@5aeeacorg.highwire.dtl.DTLVardef@1eb96ceorg.highwire.dtl.DTLVardef@1f9b899_HPS_FORMAT_FIGEXP M_FIG Building on previous work of neutrophil function we propose 3 main phases of early neutrophil recruitment and behaviour in the airways during RSV infection. Phase 1. Initial chemotaxis and adherence: Here unstimulated circulating neutrophils expressing baseline levels of CD11b migrate across infected AECs in response to chemotactic signals in the apical supernatant. Some neutrophils remain adherent to the infected AECs. Phase 2: Activation and reverse migration: once on the apical side of the epithelium, neutrophils increase expression of CD11b and other activation associated markers, and some activated neutrophils undergo reverse migration. Neutrophils with greater expression of CD11b are detected on the basolateral side Phase 3: Amplified chemotaxis and clustering: after 20 minutes, adherent neutrophils begin to rapidly cluster on RSV infected primary airway epithelial cells cultures, mediated by signalling from a dying neutrophil. Drawing created using BioRender.com. C_FIG

immunology↗

bioRxiv2021

Characterisation of B.1.1.7 and Pangolin coronavirus spike provides insights on the evolutionary trajectory of SARS-CoV-2

The recent emergence of SARS-CoV-2 variants with increased transmission, pathogenesis and immune resistance has jeopardised the global response to the COVID-19 pandemic. Determining the fundamental biology of viral variants and understanding their evolutionary trajectories will guide current mitigation measures, future genetic surveillance and vaccination strategies. Here we examine virus entry by the B.1.1.7 lineage, commonly referred to as the UK/Kent variant. Pseudovirus infection of model cell lines demonstrate that B.1.1.7 entry is enhanced relative to the Wuhan-Hu-1 reference strain, particularly under low expression of receptor ACE2. Moreover, the entry characteristics of B.1.1.7 were distinct from that of its predecessor strain containing the D614G mutation. These data suggest evolutionary tuning of spike protein function. Additionally, we found that amino acid deletions within the N-terminal domain (NTD) of spike were important for efficient entry by B.1.1.7. The NTD is a hotspot of diversity across sarbecoviruses, therefore, we further investigated this region by examining the entry of closely related CoVs. Surprisingly, Pangolin CoV spike entry was 50-100 fold enhanced relative to SARS-CoV-2; suggesting there may be evolutionary pathways by which SARS-CoV-2 may further optimise entry. Swapping the NTD between Pangolin CoV and SARS-CoV-2 demonstrates that changes in this region alone have the capacity to enhance virus entry. Thus, the NTD plays a hitherto unrecognised role in modulating spike activity, warranting further investigation and surveillance of NTD mutations.

microbiology↗

bioRxiv2020

An Entropic Safety Catch Controls Hepatitis C Virus Entry and Antibody Resistance.

E1 and E2 (E1E2), the entry proteins of Hepatitis C Virus (HCV), are unlike that of any other virus yet described, and the detailed molecular mechanisms of HCV entry/fusion remain unknown. Hypervariable region-1 (HVR-1) of E2 is a putative intrinsically disordered protein tail. Here, we demonstrate that HVR-1 has an autoinhibitory function that suppresses the activity of E1E2 on free virions; this is dependent on its conformational entropy. Crucially, to allow entry, this mechanism is turned off by host receptor interactions at the cell surface. Thus, HVR-1 is akin to a safety catch on E1E2 activity. Mutations that reduce conformational entropy in HVR-1, or genetic deletion of HVR-1, turn off the safety catch to generate hyper-reactive HCV that exhibits enhanced virus entry but is thermally unstable and acutely sensitive to neutralising antibodies. Therefore, the HVR-1 safety catch controls the efficiency of virus entry and maintains resistance to neutralising antibodies.

molecular biology↗

bioRxiv2019

Conformational switching of CD81 controls its function as a receptor for hepatitis C virus

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational opening of the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol sensing mechanism; however, the relevance of this for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81-cholesterol association, but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol unbound) or closed (cholesterol bound) conformation. The open mutant of CD81 exhibited reduced receptor activity whereas the closed mutant enhanced activity. These data are consistent with CD81 cholesterol sensing resulting in a switch between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry and CD81s function as a molecular scaffold; these insights are relevant to CD81s varied roles in both health and disease.

molecular biology↗