Biology subjects

Vahey, M. D.

Publications and source records attributed to Vahey, M. D..

13 recordsLinked to original sources

bioRxiv2025

Ciliated cells promote high infectious potential of influenza A virus through the efficient intracellular activation of hemagglutinin

Influenza viruses utilize host proteases to activate the viral fusion protein, hemagglutinin (HA), into its fusion-competent form. Although proteolytic activation of HA is essential for virus replication, the cell-type dependence of HA activation within the airway epithelium and the subcellular location(s) in which it occurs are not well-established. To address these questions, we investigated the proteolytic activation of HA in differentiated human airway epithelial cells using contemporary and historical H1N1 and H3N2 strains. We find that activation is efficient across viral strains and subtypes but depends on cellular tropism, with ciliated cells activating HA more effectively than non-ciliated cells. Similar to prior observations in immortalized cell lines, we find that HA activation occurs intracellularly, constraining the antiviral activity of host-directed protease inhibitors. These results establish that HA activation within the airway epithelium depends on cellular tropism, and identify important considerations for the development of protease inhibitors as antivirals. ImportanceInfluenza entry requires the proteolytic activation of the viral fusion protein, hemagglutinin (HA). Activation occurs as new viruses are produced by infected cells. Efficient proteolytic activation is critical for viral pathogenesis, and inhibiting the requisite proteases could provide an effective host-directed antiviral strategy. To understand cellular constraints on HA activation and its sensitivity to inhibitors, we use complementary approaches to investigate these processes in differentiated airway epithelial cells. We find that ciliated cells activate HA with higher efficiency than non-ciliated cell types, establishing a new mechanism through which cellular tropism and virus infectious potential are connected. We also establish that HA activation begins in the Golgi, which may contribute to the limited the efficacy of inhibitors we observe despite their high in vitro potency.

microbiology↗

bioRxiv2024

Antigen flexibility supports the avidity of hemagglutinin-specific antibodies at low antigen densities

The receptor-binding protein of influenza A virus, hemagglutinin (HA), is the most abundant protein on the viral surface. While high densities of HA are thought to improve cellular attachment by increasing avidity for the viral receptor, they may also increase the avidity of neutralizing antibodies. The tradeoff between these two competing effects of avidity is not well understood. To better understand how features of the viral surface influence antibody avidity, we developed fluorescence-based assays to measure dissociation kinetics and steady-state binding of antibodies to intact virions. Focusing on two antibodies that bind to the HA head domain (S139/1 and C05), we confirm that binding orientations that favor bivalent attachment of antibodies to the viral surface can offset weak monovalent affinity by facilitating crosslinking. By modulating HA density in both engineered viruses and synthetic nanoparticles, we find that bivalent antibody binding remains resilient down to one-tenth the HA density on the viral surface and, in the case of C05, that antibody occupancy increases at these lowest densities. Finally, using a combination of structure-guided modeling and antibodies that lock HA in a tilted conformation, we identify flexibility of the HA ectodomain as an additional determinant of antibody avidity. Together, these results establish features of the viral surface that help support or suppress the binding of neutralizing antibodies.

biophysics↗

bioRxiv2024

Modulation of B cell receptor activation by antibody competition

During repeated virus exposure, pre-existing antibodies can mask viral epitopes by competing with B cell receptors for antigen. Although this phenomenon has the potential to steer B cell responses away from conserved epitopes, the factors that influence epitope masking by competing antibodies remain unclear. Using engineered, influenza-reactive B cells, we investigate how antibodies influence the accessibility of epitopes on the viral surface. We find that membrane-proximal epitopes on influenza hemagglutinin are fundamentally at a disadvantage for B cell recognition because they can be blocked by both directly and indirectly competing antibodies. While many influenza-specific antibodies can inhibit B cell activation, the potency of masking depends on proximity of the targeted epitopes as well as antibody affinity, kinetics, and valency. Although most antibodies are inhibitory, we identify one that can enhance accessibility of hidden viral epitopes. Together, these findings establish rules for epitope masking that could help advance immunogen design.

immunology↗

bioRxiv2024

Genetically recoding respiratory syncytial virus to visualize nucleoprotein dynamics and virion assembly

RNA viruses possess small genomes encoding a limited repertoire of essential and often multifunctional proteins. Although genetically tagging viral proteins provides a powerful tool for dissecting mechanisms of viral replication and infection, it remains a challenge. Here, we leverage genetic code expansion to develop a recoded strain of respiratory syncytial virus (RSV) in which the multifunctional nucleoprotein is site-specifically modified with an unnatural amino acid. The resulting virus replicates exclusively in cells capable of amber stop codon suppression and is amenable to labeling with tetrazine-modified fluorophores, achieving high signal-to-background. We use this tool to visualize the transfer of nucleoprotein complexes from cytoplasmic condensates directly to budding viral filaments at the cell surface and to cytoplasmic compartments containing viral surface proteins, suggesting multiple pathways for viral assembly.

microbiology↗

bioRxiv2023

Antibody inhibition of influenza A virus assembly and release

Antibodies are frontline defenders against influenza virus infection, providing protection through multiple complementary mechanisms. Although a subset of monoclonal antibodies (mAbs) have been shown to restrict replication at the level of virus assembly and release, it remains unclear how potent and pervasive this mechanism of protection is, due in part to the challenge of separating this effect from other aspects of antibody function. To address this question, we developed imaging-based assays to determine how effectively a broad range of mAbs against the IAV surface proteins can specifically restrict viral egress. We find that classically neutralizing antibodies against hemagglutinin are broadly multifunctional, inhibiting virus assembly and release at concentrations one- to twenty-fold higher than the concentrations at which they inhibit viral entry. These antibodies are also capable of altering the morphological features of shed virions, reducing the proportion of filamentous particles. We find that antibodies against neuraminidase and M2 also restrict viral egress, and that inhibition by anti-neuraminidase mAbs is only partly attributable to a loss in enzymatic activity. In all cases, antigen crosslinking - either on the surface of the infected cell, between the viral and cell membrane, or both - plays a critical role in inhibition, and we are able to distinguish between these modes experimentally and through a structure-based computational model. Together, these results provide a framework for dissecting antibody multifunctionality that could help guide the development of improved therapeutic antibodies or vaccines, and that can be extended to other viral families and antibody isotypes.

microbiology↗

bioRxiv2022

Uncovering molecular grammars of intrinsically disordered regions that organize nucleolar fibrillar centers

The nucleolus is a multilayered structure. Each layer is thought to be a compositionally distinct phase, although how these phases form and interface with one another remains unclear. Using computational, proteomics, in vitro, and in vivo studies, we uncover distinct molecular grammars within intrinsically disordered regions (IDRs) of nucleolar proteins that localize to fibrillar centers (FCs) and dense fibrillar components (DFCs). FC and DFC proteins feature two distinct types of IDRs namely those with long tracts of acidic residues and those with blocks of lysines interspersed by acid-rich-regions. We find that phase separation driven by complex coacervation in mixtures of nucleolar proteins, with their distinctive IDRs, and ribosomal DNA and RNA molecules is sufficient to drive the formation of structural facsimiles of FCs and DFCs. One-Sentence SummaryFacsimiles of core nucleolar substructures were reconstituted via phase separation of key protein and nucleic acid mixtures.

biophysics↗

bioRxiv2022

Neuraminidase activity modulates cellular co-infection during influenza A virus multicycle growth

Infection of individual cells by multiple virions plays critical roles in the replication and spread of many viruses, but mechanisms that control cellular co-infection during multi-cycle viral growth remain unclear. Here, we investigate virus-intrinsic factors that control cellular co-infection by influenza A virus (IAV). Using quantitative fluorescence to track the spread of virions from single infected cells, we identify the IAV surface protein neuraminidase (NA) as a key determinant of cellular co-infection. We map this effect to NAs ability to deplete viral receptors from both infected and neighboring uninfected cells. In cases where viral infectious potential is low, genetic or pharmacological inhibition of NA increases the local spread of infection by increasing the viral load received by neighboring cells. These results identify virus-intrinsic factors that contribute to cellular multiplicity of infection, and suggest that optimal levels of NA activity depend on the infectious potential of the virus in question.

microbiology↗

bioRxiv2022

Six-Dimensional Single-Molecule Imaging with Isotropic Resolution using a Multi-View Reflector Microscope

We report a radially and azimuthally polarized multi-view reflector (raMVR) microscope for precise imaging of the 3D positions and 3D orientations of single molecules (SMs, 10.9 nm and 2.0{degrees} precisions using 5000 photons). These precisions are [~]1.5 times better than those of existing methods for SM orientation-localization microscopy. The raMVR microscope achieves 6D super-resolution imaging of Nile red (NR) molecules transiently bound to 150 nm, 350 nm, and 1 {micro}m-diameter lipid-coated spheres, accurately resolving their spherical morphology despite refractive-index mismatch. Simply by observing the rotational dynamics o raMVR images also resolve the infiltration of lipid membranes by amyloid-beta oligomers without covalent labeling. Finally, we demonstrate 6D imaging of HEK-293T cell membranes, where the orientations of merocyanine 540 molecules reveal heterogeneities in membrane fluidity. With its [~]2 {micro}m depth range, nearly isotropic 3D spatial resolution, and superior orientation measurement precision, we expect the raMVR microscope to enable 6D imaging of molecular dynamics within biological and chemical systems with unprecedented detail.

biophysics↗

bioRxiv2021

A morphological transformation in respiratory syncytial virus leads to enhanced complement activation

The complement system is a critical host defense against infection, playing a protective role that can also enhance disease if misregulated. Although many consequences of complement activation during viral infection are well-established, specific mechanisms that contribute to activation by different human viruses remain elusive. Here, we investigate complement activation by human respiratory syncytial virus (RSV), a respiratory pathogen that causes severe disease in infants, the immunocompromised, and the elderly. Using a strain of RSV harboring tags on the surface glycoproteins F and G, we were able to monitor opsonization of single RSV particles with monoclonal antibodies and complement components using fluorescence microscopy. These experiments revealed an antigenic hierarchy in complement activation, where antibodies that bind towards the apex of F in either the pre- or postfusion conformation are able to activate complement whereas other antibodies are not. Additionally, among antibodies that were able to activate complement, we observed preferential targeting of a subset of particles with globular morphology, in contrast to the more prevalent viral filaments. We found that enhanced complement activation on these particles arises from changes in surface curvature that occur when the viral matrix detaches from the surrounding membrane. This transformation occurs naturally over time under mild conditions, and correlates with the accumulation of postfusion F on the viral surface. Collectively, these results identify antigenic and biophysical characteristics of virus particles that contribute to the formation of immune complexes, and suggest models for how these factors may shape disease severity and adaptive immune responses to RSV.

microbiology↗

bioRxiv2020

Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion

Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene (ISG) that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an ISG screen against VSV-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of virus replication. Mechanistically, internalized 25HC accumulates in the late endosomes and blocks cholesterol export, thereby restricting SARS-CoV-2 spike protein catalyzed membrane fusion. Our results highlight a unique antiviral mechanism of 25HC and provide the molecular basis for its possible therapeutic development.

microbiology↗

bioRxiv2019

Influenza A virus surface proteins are organized to help penetrate host mucus

Influenza A virus (IAV) enters cells by binding to sialic acid on the cell surface. To accomplish this while avoiding immobilization by sialic acid in host mucus, viruses rely on a balance between the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Although genetic aspects of this balance are well-characterized, little is known about how the spatial organization of these proteins in the viral envelope may contribute. Using site-specific fluorescent labeling and super-resolution microscopy, we show that HA and NA are asymmetrically distributed on the surface of filamentous viruses, creating an organization of binding and cleaving activities that causes viruses to step consistently away from their NA-rich pole. This Brownian ratchet-like diffusion produces persistent directional mobility that resolves the viruss conflicting needs to both penetrate mucus and stably attach to the underlying cells, and could contribute to the prevalence of the filamentous phenotype in clinical isolates of IAV.

microbiology↗

bioRxiv2018

Low fidelity assembly of influenza A virus promotes escape from host cells

Influenza viruses inhabit a wide range of host environments using a limited repertoire of protein components. Unlike viruses with stereotyped shapes, influenza produces virions with significant morphological variability even within clonal populations. Whether this tendency to form pleiomorphic virions is coupled to compositional heterogeneity and whether it affects replicative fitness remains unclear. Here we address these questions by developing live strains of influenza A virus amenable to rapid compositional characterization through quantitative, site-specific labeling of viral proteins. Using these strains, we find that influenza A produces virions with broad variations in size and composition from even single infected cells. The virus leverages this phenotypic variability to survive environmental challenges including temperature changes and anti-virals. Complimenting genetic adaptations that act over larger populations and longer times, this low fidelity assembly of influenza A virus allows small populations to survive environments that fluctuate over individual replication cycles.

microbiology↗

bioRxiv2018

Claudin-4 Reconstituted in Unilamellar Vesicles is Sufficient to Form Tight Interfaces that Partition Membrane Proteins

Tight junctions have been hypothesized to act as molecular fences in the plasma membrane of epithelial cells, helping to form differentiated apical and basolateral domains. While this fence function is believed to arise from the interaction of four-pass transmembrane claudins, the complexity of tight junctions has made direct evidence of their role as a putative diffusion barrier difficult to obtain. Here we address this challenge by reconstituting claudin-4 into giant unilamellar vesicles using microfluidic jetting. We find that reconstituted claudin-4 is sufficient to form adhesive interfaces between unilamellar vesicles without accessory proteins present in vivo. By controlling the molecular composition of the inner and outer leaflets of jetted membranes, we show that claudin-4-mediated interfaces can drive partitioning of extracellular membrane proteins but not of inner or outer leaflet lipids. Our findings indicate that homotypic interactions of claudins and their small size can contribute to the polarization of epithelial cells.

cell biology↗