The entry process of viruses into host cells is complex and involves stable but transient multivalent interactions with different cell surface receptors. The initial contact of several viruses begins with attachment to heparan sulfate (HS) proteoglycans on the cell surface, which results in a cascade of events that end up with virus entry. The development of antiviral agents based on multivalent interactions to shield virus particles and block initial interactions with cellular receptors has attracted attention in antiviral research. Here, we designed nanogels with different degrees of flexibility based on dendritic polyglycerol sulfate to mimic cellular HS. The designed nanogels are nontoxic and broad-spectrum, can multivalently interact with viral glycoproteins, shield virus surfaces, and efficiently block infection. We also visualized virus-nanogel interactions as well as the uptake of nanogels by the cells through clathrin-mediated endocytosis using confocal microscopy. As many human viruses attach to the cells through HS moieties, we introduce our flexible nanogels as robust inhibitors for these viruses.

Schematic representation of synergistic action of electrostatic interactions of polyglycerol sulfate and conjugated aliphatic chains to the surface of nG-PGS.

No abstract

The influenza A virus infects target cells through multivalent interactions of its major spike proteins, hemagglutinin (HA) and neuraminidase (NA), with the cellular receptor sialic acid (SA). HA is known to mediate the attachment of the virion to the cell, whereas NA enables the release of newly formed virions by cleaving SA from the cell. Because both proteins target the same receptor but have antagonistic functions, virus infection depends on a properly tuned balance of the kinetics of HA and NA activities for viral entry to and release from the host cell. Here, dynamic single-molecule force spectroscopy, based on scanning force microscopy, was employed to determine these bond-specific kinetics, characterized by the off rate k off , rupture length x b and on rate k on , as well as the related free-energy barrier DG and the dissociation constant K D . Measurements were conducted using surface-immobilized HA and NA of the influenza A virus strain A/California/04/2009 and a novel, to our knowledge, synthetic SA-displaying receptor for functionalization of the force probe. Single-molecule force spectroscopy at force loading rates between 100 and 50,000 pN/s revealed most probable rupture forces of the protein-SA bond in the range of 10-100 pN. Using an extension of the widely applied Bell-Evans formalism by Friddle, De Yoreo, and co-workers, it is shown that HA features a smaller x b , a larger k off and a smaller DG than NA. Measurements of the binding probability at increasing contact time between the scanning force microscopy force probe and the surface allow an estimation of K D , which is found to be three times as large for HA than for NA. This suggests a stronger interaction for NA-SA than for HA-SA. The biological implications in regard to virus binding to the host cell and the release of new virions from the host cell are discussed.

Multivalency is a key principle in reinforcing reversible molecular interactions through the formation of multiple bonds. The influenza A virus deploys this strategy to bind strongly to cell surface receptors. We performed single-molecule force spectroscopy (SMFS) to investigate the rupture force required to break individual and multiple bonds formed between synthetic sialic acid (SA) receptors and the two principal spike proteins of the influenza A virus (H3N2): hemagglutinin (H3) and neuraminidase (N2). Kinetic parameters such as the rupture length (χ β ) and dissociation rate (k off ) are extracted using the model by Friddle, De Yoreo, and Noy. We found that a monovalent SA receptor binds to N2 with a significantly higher bond lifetime (270 ms) compared to that for H3 (36 ms). By extending the single-bond rupture analysis to a multibond system of n proteinreceptor pairs, we provide an unprecedented quantification of the mechanistic features of multivalency between H3 and N2 with SA receptors and show that the stability of the multivalent connection increases with the number of bonds from tens to hundreds of milliseconds. Association rates (k on ) are also provided, and an estimation of the dissociation constants (K D ) between the SA receptors to both proteins indicate a 17-fold higher binding affinity for the SA−N2 bond with respect to that of SA−H3. An optimal designed multivalent SA receptor showed a higher binding stability to the H3 protein of the influenza A virus than to the monovalent SA receptor. Our study emphasizes the influence of the scaffold on the presentation of receptors during multivalent binding.