Erratum:Influenza as a molecular walker (Chemical Science (2020) 11 (27–36) DOI: 10.1039/c9sc05149j)

The authors regret that incorrect details were given for ref. 70 in the original article. The correct version of ref. 70 is given below as ref. 1. The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.</p

Induced Förster resonance energy transfer by encapsulation of DNA-scaffold based probes inside a plant virus based protein cage

Insight into the assembly and disassembly of viruses can play a crucial role in developing cures for viral diseases. Specialized fluorescent probes can benefit the study of interactions within viruses, especially during cell studies. In this work, we developed a strategy based on Förster resonance energy transfer (FRET) to study the assembly of viruses without labeling the exterior of viruses. Instead, we exploit their encapsulation of nucleic cargo, using three different fluorescent ATTO dyes linked to single-stranded DNA oligomers, which are hybridised to a longer DNA strand. FRET is induced upon assembly of the cowpea chlorotic mottle virus, which forms monodisperse icosahedral particles of about 22 nm, thereby increasing the FRET efficiency by a factor of 8. Additionally, encapsulation of the dyes in virus-like particles induces a two-step FRET. When the formed constructs are disassembled, this FRET signal is fully reduced to the value before encapsulation. This reversible behavior makes the system a good probe for studying viral assembly and disassembly. It, furthermore, shows that multi-component supramolecular materials are stabilized in the confinement of a protein cage.</p

Receptor Density-Dependent Motility of Influenza Virus Particles on Surface Gradients

Influenza viruses can move across the surface of host cells while interacting with their glycocalyx. This motility may assist in finding or forming locations for cell entry and thereby promote cellular uptake. Because the binding to and cleavage of cell surface receptors forms the driving force for the process, the surface-bound motility of influenza is expected to be dependent on the receptor density. Surface gradients with gradually varying receptor densities are thus a valuable tool to study binding and motility processes of influenza and can function as a mimic for local receptor density variations at the glycocalyx that may steer the directionality of a virus particle in finding the proper site of uptake. We have tracked individual influenza virus particles moving over surfaces with receptor density gradients. We analyzed the extracted virus tracks first at a general level to verify neuraminidase activity and subsequently with increasing detail to quantify the receptor density-dependent behavior on the level of individual virus particles. While a directional bias was not observed, most likely due to limitations of the steepness of the surface gradient, the surface mobility and the probability of sticking were found to be significantly dependent on receptor density. A combination of high surface mobility and high dissociation probability of influenza was observed at low receptor densities, while the opposite occurred at higher receptor densities. These properties result in an effective mechanism for finding high-receptor density patches, which are believed to be a key feature of potential locations for cell entry.</p

Receptor Density-Dependent Motility of Influenza Virus Particles on Surface Gradients

Influenza viruses can move across the surface of host cells while interacting with their glycocalyx. This motility may assist in finding or forming locations for cell entry and thereby promote cellular uptake. Because the binding to and cleavage of cell surface receptors forms the driving force for the process, the surface-bound motility of influenza is expected to be dependent on the receptor density. Surface gradients with gradually varying receptor densities are thus a valuable tool to study binding and motility processes of influenza and can function as a mimic for local receptor density variations at the glycocalyx that may steer the directionality of a virus particle in finding the proper site of uptake. We have tracked individual influenza virus particles moving over surfaces with receptor density gradients. We analyzed the extracted virus tracks first at a general level to verify neuraminidase activity and subsequently with increasing detail to quantify the receptor density-dependent behavior on the level of individual virus particles. While a directional bias was not observed, most likely due to limitations of the steepness of the surface gradient, the surface mobility and the probability of sticking were found to be significantly dependent on receptor density. A combination of high surface mobility and high dissociation probability of influenza was observed at low receptor densities, while the opposite occurred at higher receptor densities. These properties result in an effective mechanism for finding high-receptor density patches, which are believed to be a key feature of potential locations for cell entry

Hierarchical Multivalent Effects Control Influenza Host Specificity

Understanding how emerging influenza viruses recognize host cells is critical in evaluating their zoonotic potential, pathogenicity, and transmissibility between humans. The surface of the influenza virus is covered with hemagglutinin (HA) proteins that can form multiple interactions with sialic acid-terminated glycans on the host cell surface. This multivalent binding affects the selectivity of the virus in ways that cannot be predicted from the individual receptor-ligand interactions alone. Here, we show that the intrinsic structural and energetic differences between the interactions of avian- or human-type receptors with influenza HA translate from individual site affinity and orientation through receptor length and density on the surface into virus avidity and specificity. We introduce a method to measure virus avidity using receptor density gradients. We found that influenza viruses attached stably to a surface at receptor densities that correspond to a minimum number of approximately 8 HA-glycan interactions, but more interactions were required if the receptors were short and human-type. Thus, the avidity and specificity of influenza viruses for a host cell depend not on the sialic acid linkage alone but on a combination of linkage and the length and density of receptors on the cell surface. Our findings suggest that threshold receptor densities play a key role in virus tropism, which is a predicting factor for both their virulence and zoonotic potential.Fil: Overeem, Nico J.. University of Twente; Países BajosFil: Hamming, P. H. Erik. University of Twente; Países BajosFil: Grant, Oliver C.. University of Georgia; Estados UnidosFil: Di Iorio, Daniele. University of Twente; Países BajosFil: Tieke, Malte. Utrecht University; Países BajosFil: Bertolino, María Candelaria. University of Twente; Países Bajos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Li, Zeshi. Utrecht University; Países BajosFil: Vos, Gaël. Utrecht University; Países BajosFil: de Vries, Robert P.. Utrecht University; Países BajosFil: Woods, Robert J.. University of Georgia; Estados UnidosFil: Tito, Nicholas B.. Electric Ant Laboratory; Países BajosFil: Boons, Geert-Jan P. H.. Utrecht University; Países BajosFil: van der Vries, Erhard. Utrecht University; Países BajosFil: Huskens, Jurriaan. University of Twente; Países Bajo

Receptor Density-Dependent Motility of Influenza Virus Particles on Surface Gradients

Influenza viruses can move across the surface of host cells while interacting with their glycocalyx. This motility may assist in finding or forming locations for cell entry and thereby promote cellular uptake. Because the binding to and cleavage of cell surface receptors forms the driving force for the process, the surface-bound motility of influenza is expected to be dependent on the receptor density. Surface gradients with gradually varying receptor densities are thus a valuable tool to study binding and motility processes of influenza and can function as a mimic for local receptor density variations at the glycocalyx that may steer the directionality of a virus particle in finding the proper site of uptake. We have tracked individual influenza virus particles moving over surfaces with receptor density gradients. We analyzed the extracted virus tracks first at a general level to verify neuraminidase activity and subsequently with increasing detail to quantify the receptor density-dependent behavior on the level of individual virus particles. While a directional bias was not observed, most likely due to limitations of the steepness of the surface gradient, the surface mobility and the probability of sticking were found to be significantly dependent on receptor density. A combination of high surface mobility and high dissociation probability of influenza was observed at low receptor densities, while the opposite occurred at higher receptor densities. These properties result in an effective mechanism for finding high-receptor density patches, which are believed to be a key feature of potential locations for cell entry

Time-Dependent Binding of Molecules and Nanoparticles at Receptor-Modified Supported Lipid Bilayer Gradients in a Microfluidic Device

Microfluidic devices are widely used for the sensing of small quantities of analytes. In these applications, the measurement can be easily perturbed by loss of analyte due to binding of the analyte outside the sensing area. We studied the binding of small molecules and nanoparticles up to 400 nm in a state-of-the-art sensing platform – receptor gradients on supported lipid bilayers (SLBs) – in a microfluidic device over time. Biotin-streptavidin was used as the model interaction motif for specific binding and a biotin-modified dye, which can bind to the streptavidin on the SLB, as a small-molecule model analyte. We used finite element simulations to show that the time-dependent binding of analytes in the sensing area depends strongly on the extent of the nonspecific binding of the vesicles, used in a preceding step to make the SLB platform, outside of the sensing area (e. g., in the tubing). At sufficiently high flow rates, proteins and nanoparticles were only partially depleted by nonspecifically adsorbed lipids, and no delayed onset of binding was observed, because of their lower diffusion coefficients. As a practical solution, a flow cell with two inlets was used to avoid the presence of nonspecifically adsorbed receptors in the sample inlet, which allowed us to decouple the formation of the sensor layer on the surface from the subsequent sensing event. We found that in the absence of lipids adsorbed to the tubing, the nonspecific binding of dye molecules was negligible

Influenza as a molecular walker

The surface of the influenza virus is decorated with the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving enzyme neuraminidase (NA). HA is responsible for host cell recognition, while NA prevents aggregation and entrapment, but the intricate mechanism of how the functions of these glycoproteins cooperate and how they are regulated by mutational responses to environmental pressures remains unclear. Recently, several groups have described the motion of influenza over surfaces and reported that this motion is inhibited by NA inhibitors. We argue that the motion of influenza resembles the motility of artificial receptor-cleaving particles called "molecular spiders". The cleaving of receptors by this type of molecular walkers leads to self-avoiding motion across a surface. When the binding and cleaving rates of molecular spiders are balanced, they move both rapidly and efficiently. The studies of molecular spiders offer new insights into the functional balance of HA and NA, but they do not address the asymmetric distribution of HA and NA on the surface of influenza. We propose that receptor-cleaving molecular walkers could play an important role in the further investigation of the motility of influenza viruses

Time‐Dependent Binding of Molecules and Nanoparticles at Receptor‐Modified Supported Lipid Bilayer Gradients in a Microfluidic Device

Microfluidic devices are widely used for the sensing of small quantities of analytes. In these applications, the measurement can be easily perturbed by loss of analyte due to binding of the analyte outside the sensing area. We studied the binding of small molecules and nanoparticles up to 400 nm in a state-of-the-art sensing platform – receptor gradients on supported lipid bilayers (SLBs) – in a microfluidic device over time. Biotin-streptavidin was used as the model interaction motif for specific binding and a biotin-modified dye, which can bind to the streptavidin on the SLB, as a small-molecule model analyte. We used finite element simulations to show that the time-dependent binding of analytes in the sensing area depends strongly on the extent of the nonspecific binding of the vesicles, used in a preceding step to make the SLB platform, outside of the sensing area (e. g., in the tubing). At sufficiently high flow rates, proteins and nanoparticles were only partially depleted by nonspecifically adsorbed lipids, and no delayed onset of binding was observed, because of their lower diffusion coefficients. As a practical solution, a flow cell with two inlets was used to avoid the presence of nonspecifically adsorbed receptors in the sample inlet, which allowed us to decouple the formation of the sensor layer on the surface from the subsequent sensing event. We found that in the absence of lipids adsorbed to the tubing, the nonspecific binding of dye molecules was negligible

A Dynamic, Supramolecular View on the Multivalent Interaction between Influenza Virus and Host Cell

Understanding how influenza viruses traverse the mucus and recognize host cells is critical for evaluating their zoonotic potential, and for prevention and treatment of the disease. The surface of the influenza A virus is covered with the receptor-binding protein hemagglutinin and the receptor-cleaving enzyme neuraminidase, which jointly control the interactions between the virus and the host cell. These proteins are organized in closely spaced trimers and tetramers to facilitate multivalent interactions with sialic acid-terminated glycans. This review shows that the individually weak multivalent interactions of influenza viruses allow superselective binding, virus-induced recruitment of receptors, and the formation of dynamic complexes that facilitate molecular walking. Techniques to measure the avidity and receptor specificity of influenza viruses are reviewed, and the pivotal role of multivalent interactions with their emergent properties in crossing the mucus and entering host cells is discussed. A model is proposed for the initiation of cell entry through virus-induced receptor clustering. The multivalent interactions of influenza viruses are maintained in a dynamic regime by a functional balance between binding and cleaving