Biology subjects

Fontana, J.

Publications and source records attributed to Fontana, J..

At least 19 recordsLinked to original sources

bioRxiv2026

The solid tumor microenvironment changes the hierarchy of CD155 and CD112 receptors, shaping checkpoint blockade outcome

Reproducing a physiologically relevant tumor microenvironment in vitro is essential for developing effective immunotherapeutic treatments. By integrating the use of combinatorial receptor blockade and organoid models we provide a deep functional understanding of CD155 and CD112 receptors in solid tumors and their impact on cellular immunotherapy and infiltration. CD226 showed plasticity in response to the environment, being able to switch between CD155 and CD112 depending on the ligand availability. In addition, CD226 drove NK cell infiltration into tumor tissues via CD155 and CD112 ligation, with CD226-CD112 interaction specifically promoting migration from the periphery to the core. Downregulation of CD155 and TIGIT induced by the tumor microenvironment and previous drug exposure reduced the long-term efficacy of TIGIT blockade. Taken together, our findings point towards using CD112R blockade in primary tumors to simultaneously enhance NK cell killing activity and promote infiltration into the tumor core via CD226 and CD112 interaction. ONE SENTENCE SUMMARYTumors shape the hierarchy of CD155-CD112 receptors, reducing TIGIT blockade efficacy, while CD226 drives NK infiltration and shows binding plasticity

immunology↗

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

Constructive neutral evolution explains the emergence of specialised ribosomes in diverse eukaryotes.

Throughout eukaryotic evolution, the structure of the ribosome has been highly conserved, featuring 80 common protein gene families. However, in many eukaryotes, paralogs of these proteins are present. "Specialised ribosomes" have been documented across diverse groups of eukaryotes where they play an important role in the regulation of translation of specific mRNAs. In the case of specialised ribosomes it has been documented that assembled ribosomes that contain specific paralogs can directly affect translational output. This has been proposed to contribute to the regulation of complex responses to environmental change and to coordinate cell-type specific physiology. This poses the question of whether ribosome specialisation principally emerges under an adaptive or neutral model of evolution. Using gene tree-species tree reconciliation, we test competing hypotheses regarding the evolutionary drivers of ribosome specialisation. We determine that examples of specialisation tend to emerge by independent duplication of the same ribosomal proteins in different lineages. We show that pathways to specialisation through paralog formation have arisen independent of: (i) paralog location within the 3D ribosome complex, and (ii) positive selection in these paralogs. We determine that the generalisable model of best fit for the evolution of paralog-mediated eukaryotic ribosomal specialisation is one of constructive neutral evolution. In lineages with small effective population sizes and increased complexity, the emergence and retention of ribosomal protein paralogs has provided the raw material for ratcheting and the emergence of translational regulation at the level of the ribosome.

evolutionary 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↗

bioRxiv2025

Unveiling the unique interaction mechanism of herpes simplex virus 2 glycoprotein C with C3b

AbstractThe complement cascade is part of the first line of defence against viral infections, and many viruses have evolved to block it. For example, glycoprotein C (gC) from Herpes Simplex Virus 1 and 2 (gC1 and gC2) facilitates infection by modulating the complement cascade through an interaction with C3b. gC is also involved in attachment and other viral processes. However, our understanding of the molecular mechanisms of gC have been limited due to the absence of a structure. AlphaFold predicts that gC contains a disordered N-terminus and three immunoglobulin-like domains. Here, we generated various gC2 constructs and demonstrated that gC2 domains 1 and 2 are necessary and sufficient to interact with C3b and block the alternative pathway. A gC2 construct lacking the N-terminus in complex with C3b was characterised by cryo-EM at 3.6 [A], providing the first structure for gC2, and revealing that the interaction is predominantly driven by gC2 domain 2 and the MG8 domain of C3b. This structure was confirmed by cross-linking mass spectrometry and by using C3b-blocking antibodies that recognised gC2 linear epitopes at the interface with C3b. Overall, the gC-C3b interaction is different from other C3b-interacting partners, providing a novel mechanism to regulate the complement cascade.

molecular biology↗

bioRxiv2025

Interactions between the 2C protein of FMDV and components of the viral replication machinery are mediated by ER-derived membranes

Foot-and-mouth disease virus (FMDV) remains an ever-present threat to the economic stability of the livestock industry and global trade. Despite this, questions remain regarding the fundamental biology underpinning the replication of this virus. Here, we examine components of the FMDV replication machinery (focussing on the viral 2C protein) and investigate the conditions under which they interact. Using a novel 2C antibody in co-immunoprecipitation experiments under different conditions followed by mass spectrometry, we identify membrane-associated proteins (such as the viral proteins 2B and precursors of 3A, which are poorly-characterised proteins involved in viral replication) along with ER-associated host proteins. In addition, our analysis shows that a number of nuclear factors interact with 2C in a membrane-independent manner, potentially being co-opted to support RNA replication of the virus. Furthermore, we demonstrate that the interaction of 2C with several viral proteins (including key members of the replication machinery and viral RNA) is maintained following ultracentrifugation, suggesting that these co-sediment as part of a complex. Our data suggest that the replication complex is ER-derived and highlight several new avenues of investigation for the disruption of the FMDV lifecycle.

microbiology↗

bioRxiv2025

Calicivirus assembly and stability are mediated by the N-terminal domain of the capsid protein with the involvement of the viral genome

Caliciviruses are important human and animal pathogens that cause varying clinical signs including gastroenteritis, respiratory illness, and hepatitis. Despite the availability of numerous calicivirus structures, relatively little is known about the mechanisms of capsid assembly and stability, or about genome packaging. Here we present the atomic structure of the RHDV virion and several related non-infectious virus-like particles, determined using cryo-EM at 2.5-3.3 [A] resolution. The inherent molecular switch, responsible for the conformational flexibility of the capsid protein VP1, is located in its N-terminal arm (NTA). The NTA establishes an extensive network of interactions on the inner capsid surface that stabilizes the hexamers and pentamers. For this structural polymorphism, we show that the NTA must interact with the RNA viral genome, that is, the genomic RNA acts concomitantly with the NTA as a molecular switch. The NTA-RNA interaction leads to specific conformational states that result in two types of VP1 dimers (the basic building blocks) necessary for T=3 capsid assembly. In addition, we used atomic force microscopy (AFM) to assess whether differences in genomic RNA content influence viral properties such as capsid stiffness in physiological conditions. These analyses highlight the mechanical role of packed RNA genome in RHDV virions, as the virion capsid pentamers are strengthened by interactions of the NTA star-like structure promoted by the viral genome. These results indicate that the interactions between the NTA and the viral genome guide the conformational states of VP1 dimers, directing capsid assembly and modulating its mechanical properties. Through interference with intermediate assemblies, the NTA network promoted by the genome could be an attractive target in future antiviral strategies. Author SummaryRabbit hemorrhagic disease virus (RHDV) is a highly contagious, often fatal calicivirus that poses a significant threat to rabbit populations worldwide. Although considerable progress has been made in elucidating the structural organization of RHDV capsids at near-atomic resolution, information regarding the nucleic acid organization within these virions and its interactions with the viral capsid remains limited. Understanding these interactions is essential for identifying mechanisms of structural polymorphism of the capsid protein, as well as virion assembly and stability. We report the cryo-electron microscopy structure, at near-atomic resolution, of RHDV virions and distinct virus-like particles, together with characterization of their biophysical properties using atomic force microscopy. The N-terminal region of the capsid protein, essential for its structural polymorphism, requires its interaction with genomic RNA. The interaction between N-terminal arms and the viral genome governs the conformational states of capsid protein dimers, thereby orchestrating capsid assembly and influencing its biophysical properties. By interfering with intermediate assemblies, the NTA network promoted by the genome could constitute an attractive target in antiviral strategies for several major human calicivirus pathogens that cause acute gastroenteritis and respiratory illness, including norovirus and sapovirus.

biophysics↗

bioRxiv2025

Adhirons are efficient tools to guide antiviral ligand discovery

Adhirons are small, antibody-like proteins expressed in bacteria that bind to biological targets with high affinity. Since their discovery, Adhirons have been isolated against over 350 targets, and they can be used for many research applications. However, their therapeutic application is at present in its early stages, hence the interest in developing novel small molecules to mimic their effect. We developed a pipeline for small molecule discovery where Adhirons act as starting templates in a ligand-based virtual screening workflow. For proof of concept, we used Adhirons against two viral systems: Crimean-Congo Haemorrhagic Fever Virus and Influenza A Virus. In both cases, a hit was identified from the mimics using biophysical and cell-based assays, highlighting how Adhirons, commercial libraries and structural approaches can be combined to accelerate small molecule inhibitor discovery of viruses.

cell biology↗

bioRxiv2025

The architecture of membrane structures involved in hepatitis C virus genome replication revealed in close-to-native conditions by cryo-electron tomography

Hepatitis C virus (HCV) infection induces extensive rearrangements of host cytoplasmic membranes, leading to the formation of multiple membranous structures that facilitate RNA replication. Current knowledge of these membranous structures has largely relied on correlative light and electron microscopy (CLEM) techniques using chemical fixation and resin embedding. To overcome these limitations, cryo-preserved cells were prepared using cryo-focused ion beam (cryo-FIB) milling and cryo-ultramicrotomy. For the first time, the contents within the membranous structures have been observed in-situ using cryo-electron tomography (cryo-ET) performed on lamellae (prepared via cryo-FIB) and on ultrathin sections (prepared via cryo-ultramicrotomy) from HCV subgenomic replicon harbouring cells. Observations from 112 cryo-electron tomograms of cryo-FIB-derived samples revealed the presence of densities within the inner vesicles of a subset of single-, double-membrane vesicles (SMVs, and DMVs respectively), as well as within multi-vesicular bodies (MVBs), which might represent the viral replication machinery. Notably, this study represents the first direct visualisation of the arrangement of non-structural proteins within a multi-membrane vesicle (MMV) observed from cryo-electron microscopy of vitreous sections (CEMOVIS). The cryo-ET methodologies established here lay the groundwork for future investigations into the architecture of the HCV replication complex, leveraging advanced computational tools for deeper structural and functional analysis.

microbiology↗

bioRxiv2024

The structural organisation of pentraxin-3 and its interactions with heavy chains of inter-alpha-inhibitor regulate crosslinking of the hyaluronan matrix

Pentraxin-3 (PTX3) is an octameric protein, comprised of eight identical protomers, that has diverse functions in reproductive biology, innate immunity and cancer. PTX3 interacts with the large polysaccharide hyaluronan (HA) to which heavy chains (HCs) of the inter--inhibitor (II) family of proteoglycans are covalently attached, playing a key role in the (non-covalent) crosslinking of HC*HA complexes. These interactions stabilise the cumulus matrix, essential for ovulation and fertilisation in mammals, and are also implicated in the formation of pathogenic matrices in the context of viral lung infections. To better understand the physiological and pathological roles of PTX3 we have analysed how its quaternary structure underpins HA crosslinking via its interactions with HCs. A combination of X-ray crystallography, cryo-electron microscopy (cryo-EM) and AlphaFold predictive modelling revealed that the C-terminal pentraxin domains of the PTX3 octamer are arranged in a central cube, with two long extensions on either side, each formed from four protomers assembled into tetrameric coiled-coil regions, essentially as described by (Noone et al., 2022; doi:10.1073/pnas.2208144119). From crystallography and cryo-EM data, we identified a network of inter-protomer salt bridges that facilitate the assembly of the octamer. Small angle X-ray scattering (SAXS) validated our model for the octameric protein, including the analysis of two PTX3 constructs: a tetrameric Half-PTX3 and a construct missing the 24 N-terminal residues ({Delta}1-24-PTX3). SAXS determined a length of [~]520 [A] for PTX3 and, combined with 3D variability analysis of cryo-EM data, defined the flexibility of the N-terminal extensions. Biophysical analyses revealed that the prototypical heavy chain HC1 does not interact with PTX3 at pH 7.4, consistent with our previous studies showing that, at this pH, PTX3 only associates with HC*HA complexes if they are formed in its presence. However, PTX3 binds to HC1 at acidic pH, and can also be incorporated into pre-formed HC*HA complexes under these conditions. This provides a novel mechanism for the regulation of PTX3-mediated HA crosslinking (e.g., during inflammation), likely mediated by a pH-dependent conformational change in HC1. The PTX3 octamer was found to associate simultaneously with up to eight HC1 molecules and, thus, has the potential to form a major crosslinking node within HC*HA matrices, i.e., where the physical and biochemical properties of resulting matrices could be tuned by the HC/PTX3 composition.

biochemistry↗

bioRxiv2024

Translational activity of 80S monosomes varies dramatically across different tissues

SummaryTranslational regulation at the stage of initiation can impact the number of ribosomes translating each mRNA molecule. However, the translational activity of single 80S ribosomes (monosomes) on mRNA is less well understood, even though these 80S monosomes represent the dominant ribosomal complexes in vivo. Here, we used cryo-EM to determine the translational activity of 80S monosomes across different tissues in Drosophila melanogaster. We discovered that while head and embryo 80S monosomes are highly translationally active, testis and ovary 80S monosomes are translationally inactive. RNA-Seq analysis of head monosome- and polysome-translated mRNAs, revealed that head 80S monosomes preferentially translate mRNAs with TOP motifs, short 5-UTRs, short ORFs and are enriched for uORFs. Overall, these findings highlight that regulation of translation initiation, and that protein synthesis is mostly performed by monosomes in head and embryo, whilst polysomes are the main source of protein production in testis and ovary. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/600330v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@12881e5org.highwire.dtl.DTLVardef@6804dborg.highwire.dtl.DTLVardef@116fa63org.highwire.dtl.DTLVardef@19095d6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LID. melanogaster 80S monosomes and polysomes purified from different tissues exhibit different translational activities C_LIO_LIHead and 0-2 hr embryo 80S monosomes are highly translationally active C_LIO_LITestis and ovary 80S monosomes are translationally inactive C_LIO_LIHead 80S monosomes preferentially translate mRNAs with TOP motifs, short 5-UTRs, short ORFs and are enriched for uORFs C_LIO_LIHead polysomes preferentially translate mRNAs with neuronal functions C_LI

molecular biology↗

bioRxiv2023

Guide RNA structure design enables combinatorial CRISPRa programs for biosynthetic profiling

Engineering bacterial metabolism to efficiently produce chemicals and materials from multi-step pathways requires optimizing multi-gene expression programs to achieve enzyme balance. CRISPR-Cas transcriptional control systems are emerging as important metabolic engineering tools for programming multi-gene expression regulation. However, poor predictability of guide RNA folding can disrupt enzyme balance through unreliable expression control. We devised a set of computational parameters that can describe guide RNA folding, and we expect them to be broadly applicable across CRISPR-Cas9 systems. Here, we correlate efficacy of modified guide RNAs (scRNAs) for CRISPR activation (CRISPRa) in E. coli with a kinetic parameter describing folding rate into the active structure. This parameter also enables forward design of new scRNAs, with no observed failures in our screen. We use CRISPRa target sequences from this set to design a system of three synthetic promoters that can orthogonally activate and tune expression of chosen outputs over a >35-fold dynamic range. Independent activation tuning allows experimental exploration of a three-dimensional expression design space via a 64-member combinatorial triple-scRNA library. We apply these CRISPRa programs to two biosynthetic pathways, demonstrating production of valuable pteridine and human milk oligosaccharide products in E. coli. Profiling these design spaces indicated expression combinations producing up to 2.3-fold higher titer than that produced by maximal expression. Mapping production can also identify bottlenecks as targets for pathway redesign, improving titer of the oligosaccharide lacto-N-tetraose by 6-fold. Aided by computational scRNA efficacy prediction, the combinatorial CRISPRa strategy enables effective optimization of multi-step metabolic pathways. More broadly, the guide RNA design rules uncovered here may enable the routine design of effective multi-guide programs for a wide range of model- and data-driven applications of CRISPR gene regulation in bacterial hosts.

synthetic biology↗

bioRxiv2023

Exploiting the Affimer platform against influenza A virus

Influenza A virus (IAV) is well known for its pandemic potential. While current surveillance and vaccination strategies are highly effective, therapeutic approaches are short-lived due to the high mutation rates of IAV. Currently, monoclonal antibodies (mAbs) have emerged as a promising approach to tackle future IAV pandemics. Additionally, several antibody-like alternatives exist that aim to improve upon mAbs. Affimers, one such alternative, benefit from a short development time, high expression levels in E. coli, and complete animal-free production. Here we exploited the Affimer platform to isolate and produce specific and potent inhibitors of IAV. Starting from a monomeric version of the IAV trimeric hemagglutinin (HA) fusion protein, we isolated 12 Affimers that inhibit IAV H3 subtype infection in vitro. Two of these Affimers were characterised in detail: they exhibited binding affinities to the target H3 HA protein in the nM range and bound specifically to the HA1 head domain. Cryo-EM employing a novel spray approach to prepare cryo-grids allowed us to image HA-Affimer complexes. Combined with functional assays, we determined that the mode of inhibition of these Affimers is based on blocking the interaction of HA to the host-cell receptor - sialic acid. Additionally, these Affimers inhibited IAV strains closely related to the one employed for Affimer isolation. Overall, these results support the use of Affimers as an alternative to existing targeted therapies for IAV and pave the way for their use as diagnostic reagents. ImportanceInfluenza A virus is one of the few viruses that can cause devastating pandemics. Due to the high mutation rates of this virus, annual vaccination is required and antivirals are short-lived. Monoclonal antibodies present a promising approach to tackle influenza virus infections, but are associated with some limitations. To improve on this strategy, we explored the Affimer platform, which are antibody-like, bacterially made proteins. By performing phage-display against a monomeric version of influenza virus fusion protein, an established viral target, we were able to isolate Affimers that inhibit influenza virus infection in vitro. We characterised the mechanism of inhibition of the Affimers by challenging with related influenza virus strains. We additionally characterised an HA-Affimer complex structure, using a novel approach to prepare samples for cryo-electron microscopy. Overall, these results show that Affimers are a promising tool against influenza virus infection.

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↗

bioRxiv2022

Organisation of the orthobunyavirus tripodal spike and the structural changes induced by low pH and K+ during entry

Following internalisation, viruses employ the changing environment of maturing endosomes as cues to promote endosomal escape, a process mediated by viral glycoproteins. Specifically, we previously showed that both high [K+] and low pH promote entry of Bunyamwera virus (BUNV), the prototypical bunyavirus. Here, we used sub-tomogram averaging combined with AlphaFold, to generate a pseudo-atomic model of the whole glycoprotein envelope of BUNV. This allowed us to unambiguously locate the Gc fusion domain and its chaperone Gn within the floor domain of the spike. We also confirmed that low pH and high [K+] alters the viral glycoproteins, resulting in an activated intermediate state functionally-distinct from the highly ordered ground state, and we localize these changes to the floor domain. Biochemical data suggests that in this intermediate state the viral fusion loops are partially exposed and selectively interact with host cell membranes. Taken together, we reveal new mechanistic understanding of the requirements for virus entry.

microbiology↗

bioRxiv2021

The structure of a native orthobunyavirus ribonucleoprotein reveals a key role for viral RNA in maintaining its helical architecture

The Bunyavirales order of RNA viruses comprises emerging pathogens for which approved preventative or therapeutic measures for human use are not available. The genome of all Bunyavirales consists of negative-sense RNA segments wrapped by the virus-encoded nucleocapsid protein (NP) to form ribonucleoproteins (RNPs). RNPs represent the active template for RNA synthesis and the form in which the genome is packaged into virions, functions that require inherent flexibility. We present a pseudo-atomic model of a native RNP purified from Bunyamwera virus (BUNV), the prototypical Bunyavirales member, based on a cryo-electron microscopy (cryo-EM) average at 13 [A] resolution with subsequent fitting of the BUNV NP crystal structure by molecular dynamics. We show the BUNV RNP possesses relaxed helical architecture, with successive helical turns separated by [~]18 [A]. The model shows that adjacent NP monomers in the RNP chain interact laterally through flexible N- and C-terminal arms, with no helix-stabilizing interactions along the longitudinal axis. Instead, EM analysis of RNase-treated RNPs suggests their chain integrity is dependent on the encapsidated genomic RNA, thus providing the molecular basis for RNP flexibility. Overall, this work will assist in designing anti-viral compounds targeting the RNP and inform studies on bunyaviral RNP assembly, packaging and RNA replication. SignificanceBunyaviruses are emerging RNA viruses that cause significant disease and economic burden and for which vaccines or therapies approved for human use do not exist. The bunyavirus genome does not exist as naked RNA; instead it is wrapped up by the nucleoprotein (NP) to form a ribonucleoprotein (RNP). Using the prototypical bunyavirus, Bunyamwera virus, we determined the 3D structure of the native RNP, revealing a helical architecture with NP molecules linked by lateral contacts only, with no helix-stabilizing longitudinal contacts. Instead, the RNA genome itself plays a role in maintaining the helical architecture, allowing a high degree of flexibility that is critical for several stages of the virus replication cycle, such as segment circularization and genome packaging into virions.

microbiology↗