Biology subjects

Ivanovic, T.

Publications and source records attributed to Ivanovic, T..

5 recordsLinked to original sources

bioRxiv2026

Antibodies to influenza A virus hemagglutinin and neuraminidase limit egress and alter the physical properties of released virus particles

Influenza A virus (IAV)-specific antibodies neutralize mature virions by inhibiting functional sites or, in some cases, by promoting virion aggregation. Many antibodies also act on infected cells to reduce virion yields, but the underlying mechanisms and effects on the physical properties and function of released virus particles remain incompletely characterized. Here we use flow virometry to acquire high-sensitivity yield and size measurements of virus particles released in the presence of antibodies. Combined with digital-droplet PCR and electron microscopy, this approach enables comprehensive characterization of antibody-induced changes in particle genome-content and morphology. We show that antibodies rapidly and dynamically alter released particle distributions, reducing yields and inducing the production of larger particles. Both effects result in part from aggregation induced by crosslinking of viral antigen on the infected-cell surface and inhibition of viral NA. However, yield reduction is not fully explained by aggregation, and a subset of induced larger particles are elongated virions. Finally, particles formed in the presence of HA stem-binding antibody, which does not inhibit attachment of mature virions, show reduced attachment in subsequent rounds of infection. Altogether, we uncover an unappreciated mechanism by which antibodies interfere with viral infection that occurs only during budding and release. Our work highlights the necessity of studying how the immune response shapes virus populations in the context of active infection processes.

microbiology↗

bioRxiv2026

Influenza A virus membrane fusion is regulated by the balance between receptor binding and cleavage

Interactions between influenza A virus (IAV) and its host receptor, sialic acid, influence multiple stages of infection through the opposing activities of hemagglutinin (HA) and neuraminidase (NA). Multivalent HA binding to low-affinity receptors generates high avidity and attachment specificity, while NA-mediated receptor cleavage promotes penetration through sialylated respiratory mucus and progeny virion release. The role of receptor interactions in post-attachment entry, particularly during genome delivery through HA-mediated endosomal membrane fusion, remains unresolved due to conflicting data, limited control of receptor conditions, and difficulty separating attachment from fusion at low efficiency. Here, we address this question using a flow-cytometry based assay that quantifies time-resolved lipid mixing in hundreds of individual virion-membrane pairs per second, combined with programmable control of receptor density and chemistry on target membranes. We show that HA-receptor interactions regulate membrane fusion efficiency by promoting productive fusion-peptide insertion by HA. This effect depends on receptor context: within a defined regime, lipid-mixing efficiency increases with receptor density whereas NA activity reduces it by depleting receptors. Receptor type and HA-receptor binding avidity further modulate lipid-mixing outcomes, establishing a direct link between HA-receptor interactions and fusion efficiency. Together, these results identify receptor binding as an active regulator of membrane fusion and provide a framework that reconciles prior conflicting observations. More broadly, they extend the functional interplay between HA and NA to the level of membrane fusion, with implications for viral adaptation and host specificity. Significance StatementWe resolve a long-standing question by showing that host receptor binding by HA and cleavage by NA regulate influenza A virus membrane fusion, the entry step delivering the viral genome into the cell. HA-receptor engagement promotes productive HA membrane insertion, leading to fusion. Receptor density, HA-receptor affinity, and NA catalytic activity tune fusion outcomes. These results were enabled by two methodological advances: precise control of receptor presentation on target membranes and sensitive measurement of attachment and membrane fusion at the single-virion level. By defining how the opposing activities of HA and NA regulate fusion, this study extends their functional interplay to a key step in entry and provides new insight into how these proteins coevolve during viral adaptation.

microbiology↗

bioRxiv2023

Influenza A Virus Infections Sense Host Membrane Tension to Dynamically Tune Assembly

Enveloped viruses often exhibit a pleomorphic morphology, ranging in size from 100nm spheres to tens-of-micron long filaments. For influenza A virus (IAV), spheres enable rapid replication and minimize metabolic cost, while filaments resist effects of antibodies or other cell-entry pressures. The current paradigm is that virion shape changes require genetic adaptation; however, a virus evolved to alter its shape phenotypically would outperform one that relies on genetic selection. Using a novel quantitative flow virometry assay to characterize virion shape dynamics we find that IAV rapidly tunes its shape distribution to favor spheres under optimal, and filaments under attenuating conditions including the presence of antibodies. We identify membrane tension as a key cue sensed by IAV determining shape distributions. This phenotypic shift outpaces genetic change and serves to enable additional life cycles under pressure. Our work expands knowledge of the complex host-virus interplay to include viral responses to the local environment by optimizing its structure to maximize replication and ultimately host-host transmission.

microbiology↗

bioRxiv2022

Hemagglutinin stability determines influenza A virus susceptibility to a broad-spectrum fusion inhibitor Arbidol

Understanding mechanisms of resistance to antiviral inhibitors can reveal nuanced features of targeted viral mechanisms and, in turn, lead to improved strategies for inhibitor design. Arbidol is a broad-spectrum antiviral which binds to and prevents the fusion-associated conformational changes in the trimeric influenza hemagglutinin (HA). The rate-limiting step during HA-mediated membrane fusion is the release of the hydrophobic fusion peptides from a conserved pocket on HA. Here, we investigated how destabilizing or stabilizing mutations in or near the fusion peptide affect viral sensitivity to Arbidol. The degree of sensitivity was proportional to the extent of fusion peptide stability on the pre-fusion HA: stabilized mutants were more sensitive, and destabilized ones resistant to Arbidol. Single-virion membrane fusion experiments for representative Wild Type and mutant viruses demonstrated that resistance is a direct consequence of fusion-peptide destabilization not dependent on reduced Arbidol binding to HA at neutral pH. Our results support the model whereby the probability of individual HAs extending to engage the target membrane is determined by the composite of two critical forces: a tug on the fusion peptide by the extension of the central coiled-coil on HA, and the key interactions stabilizing fusion peptide in the pre-fusion pocket. Arbidol increases the free-energy penalty for coiled-coil extension, but destabilizing mutations decrease the free-energy cost for fusion peptide release, accounting for the observed resistance. Our findings have broad implications for fusion-inhibitor design, viral mechanisms of resistance, and our basic understanding of HA-mediated membrane fusion.

microbiology↗

bioRxiv2019

Phenotypic size heterogeneity is a viral mechanism of persistence

Assembly of many enveloped animal viruses yields a mixture of particle morphologies, from small, essentially isometric forms to vastly longer, filamentous forms. Selective advantage of pleomorphic virus structure is apparent only in vivo, hindering functional characterization of distinct particle shapes. Here we sought to mimic the in vivo pressures on virus entry in cultured cells and in single-particle experiments of membrane fusion for influenza virus preparations enriched in spherical or filamentous particles. We show that filamentous shape confers functional advantage in the presence of neutralizing antibodies or fusion inhibitors and in cases of only limited fusion-protein activation. For very long particles, inactivation of >95% of associated fusion proteins still permits enough active-protein cooperation to induce membrane merger. Experiments with Ebola virus-like particles show that resistance to antibody pressure is a conserved feature of filamentous particles. Our results offer a strategy for averting drug resistance or immune evasion by targeting filamentous virus particles.

microbiology↗