Biology subjects

Barr, J. N.

Publications and source records attributed to Barr, J. N..

11 recordsLinked to original sources

bioRxiv2026

Interaction of Bunyamwera Virus Non-Structural Protein NSm with Cellular BNIP1 is Required for Efficient Viral Gene Expression and Replication

Bunyamwera virus (BUNV) is the prototypical member of the Peribunyaviridae family of arthropod-borne viruses and possesses a genome comprising three segments of negative-sense RNA, named small, medium and large. The medium segment encodes a polyprotein that is processed to form Gn and Gc spikes and a non-structural protein, NSm. The role of NSm during replication in mammalian cells is poorly characterized, although it associates with a Golgi-derived structure called the virus factory (VF), the site of BUNV genome replication and virion assembly. To further define NSm function, we generated an epitope-tagged BUNV and used co-immunoprecipitation and quantitative proteomics to identify host interacting partners. NSm interacted with BCL-2 interacting protein 1 (BNIP1), a SNARE protein involved in COPI vesicle trafficking, with the importance of this interaction demonstrated by siRNA-mediated knockdown of BNIP1 expression, which significantly reduced BUNV gene expression and virion production. Interestingly, NSm also interacted with components of the NRZ complex, involved in COPI vesicle tethering in association with BNIP1, and inhibition of COPI complex formation resulted in loss of NSm expression. Taken together, our results identify BNIP1 as a host cell factor necessary for efficient BUNV replication and suggest the cellular localization of NSm at the VF is COPI-dependent.

microbiology↗

bioRxiv2026

Molecular dialogue between Orthonairovirus and tick: RNA-protein interactome of Hazara virus, a BSL2 model of Crimean-Congo Hemorrhagic Fever virus, in Hyalomma cells

Climate change and ecosystem collapse promote geographic expansion of vector-borne diseases, as witnessed by the recent incursions into Spain of the virus responsible for Crimean-Congo hemorrhagic fever (CCHFV). CCHFV is maintained in a tick-vertebrate cycle, principally involving ticks of the genus Hyalomma. Faced with the spread of Hyalomma ticks, and therefore the threat of a natural introduction of CCHFV into Western Europe, appropriate surveillance tools and control measures need to be implemented. It is both within and by the tick that CCHFV is maintained and spread in the environment. Despite prolonged portage of the virus, the tick is not overtly affected by CHFV infection. One of the prerequisites in conceiving control strategies is to understand the molecular mechanisms that intimately link the virus to its arthropod host. Despite the central role of the tick in the biology of CCHFV, these mechanisms are ill-defined, owing in part to the constraints associated with handling CCHFV-infected ticks in biosafety level 4 containment. In this study, we established the network of interactions between the S segment of the RNA genome Hazara virus (HAZV), a BSL-2 model of CCHFV, and Hyalomma proteins using ChIRP-MS technique. We identified 166 tick proteins, 21 of which have been described as RNA-binding proteins. Gene ontology and pathway enrichment analyses revealed that the S segment RNA interacts predominantly with mitochondrial proteins that belong to various mitochondrial metabolic pathways.

microbiology↗

bioRxiv2026

A Systematic Evaluation of Protein Phase Separation Predictors Across Diverse Protein Landscapes

BackgroundLiquid-liquid phase separation (LLPS) plays a central role in cellular regulation, with its dysregulation linked to numerous disease outcomes. LLPS has also being increasingly implicated as critical in various biological contexts, with a key example being virus replication. These findings have driven the development of numerous computational predictors to screen and identify phase-separating proteins from sequence and/or structural models. Despite the growing need for these tools, their comparative performance across diverse biological contexts remains incompletely understood, complicating tool selection and result interpretation. ResultsA systematic comparative analysis of nine LLPS prediction algorithms was conducted using multiple curated datasets comprising both LLPS-positive (LLPS+) and -negative (LLPS-) proteins. The datasets span a range of biologically relevant scenarios, including intrinsically disordered proteins, folded proteins, proteins with LLPS-abolishing variants, benchmark datasets, and viral proteins. Substantial variability in predictive performance was observed across algorithms when assessing proteins of different structural classes. Numerous showed reduced accuracy in distinguishing LLPS+ and LLPS- folded proteins. Sensitivity to the impact of small LLPS-abolishing mutations also varied markedly between predictors, with structure-informed algorithms generally outperforming most sequence-based predictors. Similar decreases in predictive capability were also observed across several algorithms when analysing viral proteins, which are often-under-represented in predictor training datasets. ConclusionsThese results demonstrate that LLPS predictor performance is strongly context dependent, leading to different predictors being optimal for different biological questions. For overall protein assessment, DeePhase and Molphase provided the most consistently accurate predictions, being the least impacted by structural bias. For assessing the impact of small mutations on LLPS propensity, PSPHunter, a built-for-purpose algorithm, reliably predicts mutation impacts with structure-informed algorithms PSPire and PICNIC also providing strong insight. Across all evaluated datasets, the findings highlight the need for well-benchmarked training and testing data that encompasses a broad and diverse range of protein classes.

bioinformatics↗

bioRxiv2026

Screening of MMV open-source libraries using Bunyamwera virus as a model reveals inhibitors of Oropouche virus infection

Arboviral infections remain a major public health concern in tropical and subtropical regions, where environmental and socioeconomic conditions facilitate the circulation of diverse RNA viruses, including over 350 members of the recently renamed Peribunyaviridae family. In this regard, Oropouche virus (OROV) has caused explosive outbreaks in the Amazon region and shows wider distribution in Brazil, with confirmed neurological infections and fatalities in 2024. The absence of effective antiviral therapies highlights an urgent need for the discovery of anti-OROV drugs. In this work we evaluated the antiviral potential of the Pandemic Response Box and Global Health Priority Box libraries from Medicines for Malaria Venture (MMV) against OROV, using a well-established Bunyamwera virus (BUNV) system, widely used as a prototype of the Peribunyaviridae family, expressing the reporter gene eGFP. A screening protocol based on fluorescent reporter gene detection by live cell imaging was employed. 9 compounds were identified with significant activity against BUNV-eGFP with 3 being highlighted as the most promising hits: Trimetrexate, GSK-983 and MMV1634385. These compounds were validated by testing for activity against OROV, and in these assays, GSK-983 exhibited the lowest EC50 value and consequently the highest selectivity index. Future studies should evaluate the antiviral efficacy of GSK-983 and assess its pharmacokinetic profile. Furthermore, our results indicate that our approach employing BUNV-eGFP as a model against OROV can lead to the discovery of promising and interesting hit compounds, thereby providing a validated screening approach for future antivirals against Oropouche fever.

microbiology↗

bioRxiv2026

Host cell potassium ion channels KCNJ2 (KIR2.1), KCNJ13 (KIR7.1) and KCNMA1 (BKCa) mediate escape of Bunyamwera virus from late endosomal compartments

The Orthobunyavirus genus within the Peribunyaviridae family of enveloped arthropod-borne negative-sense RNA viruses includes species associated with serious or fatal disease in both animals and humans such as Schmallenberg, La Crosse and Oropouche viruses. Orthobunyaviruses (OBVs) are internalised into cells by endocytosis and release their genomes following fusion with late endosomal (LE) membranes, triggered by low pH of the luminal milieu. There is mounting evidence to suggest OBV endosomal escape is also influenced by potassium ions (K+), which increase in concentration as endosomes mature. Endosomal K+ flux is controlled by cellular K+ channels, and we previously showed that K+ channel blockade using broad spectrum pharmacological inhibition abrogates OBV infection, with virions trapped within the endosomes. However, the K+ channels regulating this process are unknown. Herein, to identify the K+ channels involved, we studied Bunyamwera virus (BUNV), the prototypical OBV, and screened a siRNA library targeting 342 human ion channels. We identified 19 K+ channels whose knockdown inhibited BUNV gene expression by over 50%, with at least seven channels affecting BUNV at the entry stage, suggesting they exert a combinatorial influence. Of these seven channels, we used both pharmacological inhibition and genetic channel manipulation to show that OBV escape from CD63- and Rab7-positive LEs is controlled by KCNJ2 (KIR2.1), KCNJ13 (KIR7.1) and KCNMA1 (BKCa). These studies add to the understanding of host factors influencing OBV entry, regulation of endosomal ion flux, and could aid in the design or repurposing of therapeutics for the treatment of OBV-associated disease.

cell biology↗

bioRxiv2025

The orthobunyavirus Gc glycoprotein head and stalk drives an infectious virion assembly pathway that is specific for the insect host

The Orthobunyavirus genus of arthropod-borne segmented RNA viruses comprises several important pathogens including the human-infecting Oropouche virus and animal-infecting Schmallenberg virus (SBV). The prototypical Bunyamwera orthobunyavirus (BUNV) possesses envelope-embedded glycoprotein spikes, with Gc head and stalk domains forming distinctive tripods covering the envelope-proximal Gn. Spike ectodomains mediate virus entry, while endodomains interact with nucleoprotein (NP) enwrapped genome segments to mediate virion assembly. Interestingly, BUNV Gc head/stalk domains are redundant for virus growth in mammalian cells, consistent with isolations of SBV from animals bearing head/stalk deletions. However, orthobunyavirus isolations from arthropods in nature appear to maintain these domains strictly. To investigate this discrepancy, we compared the multiplication characteristics of wildtype BUNV (BUNV-WT) with a Gc head/stalk deleted BUNV (BUNV-{Delta}7). In mammalian cells BUNV-WT and BUNV-{Delta}7 grew to equivalent titres, whereas BUNV-{Delta}7 titres from insect cells were 1000-fold lower than BUNV-WT and strikingly produced no virions following blood meal infection of Aedes mosquitoes. To understand this insect-specific restriction in virion production, we showed the intracellular abundance of BUNV-WT and BUNV-{Delta}7 Gc and NP components were equivalent, suggesting {Delta}7-Gc was assembly-deficient. To explore this, we investigated Gc and {Delta}7-Gc interactions during BUNV-WT and BUNV-{Delta}7 infections of both insect and mammalian cells by co-immunoprecipitation and multiplex mass spectrometry, revealing {Delta}7-Gc exhibited markedly reduced NP interactions in insect cells. We propose Gc recruits genome segments during virion assembly and that the complete Gc head/stalk assembly is maintained in nature due to its essential role in the insect host. IMPORTANCEOrthobunyaviruses are arthropod-borne viruses that cause severe disease in humans and animals, including congenital malformations and abortions. Orthobunyavirus Gn and Gc glycoproteins form spikes, which mediate entry and genome recruitment during assembly. In nature, OBVs bearing large deletions within Gc head/stalk domains have been isolated from animals, yet the head/stalk domains appear to be strictly maintained within insects. To investigate this discrepancy, we compared the multiplication of Bunyamwera orthobunyavirus (BUNV-WT) with head/stalk-deleted variant (BUNV-{Delta}7). In mammalian cells both viruses reached similar titres, but strikingly BUNV-{Delta}7 failed to produce virions in both insect cells and Aedes mosquitoes. We showed this was because BUNV-{Delta}7 failed to assemble new virions, revealing its head/stalk-deleted Gc was deficient in interactions with genome components. We propose that Gc drives species-specific interactions with genome segments during virion assembly, explaining why Gc head/stalk domains are conserved in nature due to their essential role in the insect host.

microbiology↗

bioRxiv2024

Single cell variation and rapid emergence of phenotypic heterogeneity in cell lines- a cautionary tale for devotees of CRISPR-Cas9.

The use and publication of research utilising CRISPR/cas9 in gene editing and knock outs (KOs) within cell lines is now widespread and has proved extremely powerful in the interrogation of gene function. However, the potential of experimental artefacts resulting from the need to generate single cell clones post gene-manipulation has been somewhat overlooked. In this study, we show that the commonly used pulmonary cell line A549 displays significant heterogeneity in terms of their gene expression, and that individual cells from a population exhibit dramatically different susceptibility to viral infection from a range of viruses including respiratory syncytial virus (RSV), influenza A virus (IAV), Hazara virus (HAZV), lymphocytic choriomeningitis virus (LCMV) and Bunyamwera virus (BUNV). Moreover, we demonstrate the rapid re-emergence of phenotypic heterogeneity even after cloning. These results demonstrate the need for caution in interpreting results from CRISPR screens and CRISPR-KO validation studies, especially in the study of viral infection.

microbiology↗

bioRxiv2023

The cryoEM structure of the Hendra henipavirus nucleoprotein reveals insights into paramyxoviral nucleocapsid architectures

We report the first cryoEM structure of the Hendra henipavirus nucleoprotein in complex with RNA, at 3.5 [A] resolution, derived from single particle analysis of homotetradecameric RNA-bound N protein rings exhibiting D14 symmetry. The structure of the HeV N protein adopts the common bi-lobed paramyxoviral N protein fold; the N-terminal and C-terminal globular domains are bisected by an RNA binding cleft containing six RNA nucleotides and are flanked by the N-terminal and C-terminal arms, respectively. In common with other paramyxoviral nucleocapsids, the lateral interface between adjacent N and N+1 protomers involves electrostatic and hydrophobic interactions mediated primarily through the N-terminal arm and globular domains with minor contribution from the C-terminal arm. However, the HeV N multimeric assembly uniquely identifies an additional interaction between N+1 and N-1 protomers. The model presented here broadens the understanding of RNA-bound paramyxoviral nucleocapsid architectures and provides a platform for further insight into the molecular biology of HeV, as well as the development of antiviral interventions.

microbiology↗

bioRxiv2023

Lymphocytic choriomeningitis arenavirus utilises tunnelling nanotube-like intercellular connections for cell-to-cell spread

The Arenaviridae family within the Bunyavirales order of segmented RNA viruses contains over 50 species grouped into four genera, Antennavirus, Hartmanivirus, Mammarenavirus and Reptarenavirus. Several mammarenaviruses are associated with fatal hemorrhagic fevers, including Lassa, Lujo and Junin viruses. The mammarenavirus member lymphocytic choriomeningitis virus (LCMV) is largely non-pathogenic to humans and represents a tractable model system for studying arenavirus molecular and cellular biology. During infection of cells in culture, a high proportion of LCMV spread is between directly neighbouring cells. Consistent with this observation LCMV-infected cells extrude multiple tunnelling nanotube (TNT)-like structures forming intercellular connections that could provide a route of cell-to-cell spread. To investigate this, we used recombinant LCMV with engineered epitope tags in glycoprotein spike (GP-1) and matrix (Z) proteins, alongside nucleoprotein (NP) antisera, to reveal that all three major structural proteins co-localised within TNT-like connections. Furthermore, utilising fluorescent in situ hybridisation (FISH) we showed NP also co-localised with LCMV genomic sense RNA. Taken together, these observations suggested LCMV virions pass between cells through intercellular connections to infect new cells. Consistent with this, addition of a potent LCMV neutralising antibody to supernatants during infection failed to block LCMV spread through cultures, revealing that cell-to-cell connectivity plays a major role in LCMV transmission. This is the first report of cell-cell infection via TNT-like connections for any species of the 14 families within the Bunyavirales order. This study furthers our understanding of how arenaviruses manipulate the host to establish infection, which may aid in the development of effective anti-viral therapeutics. IMPORTANCEArenaviruses include some of the most serious human pathogens in existence, although no clinically approved vaccines or therapies are currently available to prevent their associated disease. As with most pathogens, transmission of arenaviruses from one cell to another is a critical aspect of infection and resulting pathogenicity. Here, we showed that model arenavirus lymphocytic choriomeningitis virus (LCMV) can spread between cells without exposure to the extracellular space. We visualized the three major LCMV structural proteins, namely nucleoprotein, glycoprotein spike and matrix co-localized along with genomic RNA within tubular structures connecting adjacent cells. The use of a potent neutralizing antibody to block the extracellular route of LCMV transmission reduced spread within cultured cells to approximately half that of untreated cultures. Taken together, these results suggest intercellular connections represent important conduits for arenavirus spread. This information will aid in the development of antiviral strategies that prevent both intra- and extracellular transmission routes.

microbiology↗

bioRxiv2023

Lymphocytic choriomeningitis arenavirus requires cellular COPI and AP-4 complexes for efficient replication and virion production.

Lymphocytic choriomeningitis virus (LCMV) is a bisegmented negative-sense RNA virus classified within the Arenaviridae family of the Bunyavirales order. LCMV is associated with fatal disease in immunocompromised populations, and as the prototypical arenavirus, acts as a model for the many serious human pathogens within the Arenaviridae family. Here, we examined the dependence of LCMV multiplication on cellular trafficking components using a recombinant LCMV expressing enhanced green fluorescent protein in conjunction with a curated siRNA library. The screen revealed a requirement for subunits of both the coat protein 1 (COPI) coatamer and adapter protein 4 (AP-4) complexes. By rescuing a recombinant LCMV harbouring a FLAG tagged GP-1 envelope spike (rLCMV-GP1-FLAG) we showed infection resulted in marked co-localization of COPI and AP-4 component with both LCMV nucleoprotein (NP) and GP-1. Time-of-addition studies using brefeldin A (BFA), an ARF-I inhibitor that prevents formation of both COPI and AP-4 complexes, suggested these cellular components were involved in late stages of the LCMV multiplication cycle. Consistent with this finding, BFA treatment at similar late time-points resulted in a marked redistribution of NP and GP-1, and subsequent loss of COPI/AP-4 co-localization. Finally, titration of released virus within supernatant of BFA-treated cells revealed a 10-fold decrease in viral titres, greater than the 2-fold BFA-mediated reduction in NP expression. Taken together, these findings suggest COPI and AP-4 complexes are important host cell factors that are required for efficient LCMV assembly and egress. ImportanceArenaviruses are rodent-borne, segmented, negative-sense RNA viruses, with several members responsible for fatal human disease, with the prototypic member LCMV being under-recognised as a pathogen capable of inflicting neurological infections with fatal outcome. Here, we assessed the impact of siRNA knockdown of host cell trafficking genes on LCMV multiplication. We reveal the requirement of host cellular COPI and AP-4 complexes for efficient LCMV multiplication, acting late in the replication cycle, at the stages of egress and assembly. Collectively, our findings improve the understanding of arenaviruses host-pathogen interactions and reveal novel cellular trafficking pathways required during infection. Moreover, this study may lead to the discovery of novel therapeutic targets for arenaviruses to prevent serious human disease.

microbiology↗

bioRxiv2023

Cellular endosomal potassium ion flux regulates arenavirus uncoating during virus entry

Lymphocytic choriomeningitis virus (LCMV) is a model arenavirus that causes fatalities within immunocompromised populations. To enter cells, the LCMV envelope fuses with endosomal membranes, for which two requirements are low pH and interaction between LCMV GP spike and receptor CD164. LCMV subsequently uncoats, where genome-associated NP separates from Z matrix. To further examine LCMV entry, an siRNA screen identified K+ channels as important for LCMV infection, and pharmacological inhibition confirmed K+ involvement during entry. We tracked incoming virions along their entry pathway under physiological conditions, where uncoating was signified by separation of NP and Z. In contrast, K+ channel blockade, prevented uncoating, trapping virions within Rab7 and CD164-positive endosomes, identifying K+ as a third LCMV entry requirement. K+ did not increase GP/CD164 binding, thus we suggest K+ mediates uncoating by modulating NP/Z interactions within the virion interior. These results suggest repurposing licensed K+ channel inhibitors represents a potential anti-arenaviral strategy.

cell biology↗