Sulfated Polyglycerol-Modified Hydrogels for Binding HSV-1 and RSV.

Heparan sulfate (HS) is a highly sulfated polysaccharide on the surface of mammalian cells and in the extracellular matrix and has been found to be important for virus binding and infection. In this work, we designed synthetic hydrogels with viral binding and deactivation activities through the postfunctionalization of an HS-mimicking polyelectrolyte and alkyl chains. Three polyglycerol-based hydrogels were prepared as substrates and postfunctionalized by sulfated linear polyglycerol (lPGS) via thiol-ene click reaction. The viral binding properties were studied using herpes simplex virus type 1 (HSV-1) and respiratory syncytial virus (RSV). The effect of hydrogel types and molecular weight (Mw) of conjugated lPGS on viral binding properties was also assessed, and promising binding activities were observed in all lPGS-functionalized samples. Further coupling of 11 carbons long alkyl chains to the hydrogel revealed virucidal properties caused by destruction of the viral envelope, as shown by atomic force microscopy (AFM) imaging.

Evaluation of Multivalent Sialylated Polyglycerols for Resistance Induction in and Broad Antiviral Activity against Influenza A Viruses.

The development of multivalent sialic acid-based inhibitors active against a variety of influenza A virus (IAV) strains has been hampered by high genetic and structural variability of the targeted viral hemagglutinin (HA). Here, we addressed this challenge by employing sialylated polyglycerols (PGs). Efficacy of prototypic PGs was restricted to a narrow spectrum of IAV strains. To understand this restriction, we selected IAV mutants resistant to a prototypic multivalent sialylated PG by serial passaging. Resistance mutations mapped to the receptor binding site of HA, which was accompanied by altered receptor binding profiles of mutant viruses as detected by glycan array analysis. Specifying the inhibitor functionalization to 2,6-α-sialyllactose (SL) and adjusting the linker yielded a rationally designed inhibitor covering an extended spectrum of inhibited IAV strains. These results highlight the importance of integrating virological data with chemical synthesis and structural data for the development of sialylated PGs toward broad anti-influenza compounds.

Quantification of Multivalent Interactions between Sialic Acid and Influenza A Virus Spike Proteins by Single-Molecule Force Spectroscopy.

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 (χß) and dissociation rate (koff) 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 protein-receptor 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 (kon) are also provided, and an estimation of the dissociation constants (KD) 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.

Force Spectroscopy Shows Dynamic Binding of Influenza Hemagglutinin and Neuraminidase to Sialic Acid.

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 koff, rupture length xß and on rate kon, as well as the related free-energy barrier ΔG and the dissociation constant KD. 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 xß, a larger koff and a smaller ΔG 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 KD, 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.

Sialyl-LacNAc-PNA⋠DNA Concatamers by Rolling-Circle Amplification as Multivalent Inhibitors of Influenza†A Virus Particles.

The surfaces of influenza A virus (IAV) particles are packed with hundreds of homo-trimeric hemagglutinins (HAs). Monovalent sugars have low affinity for HA, but distance-optimized bivalent sialyl-LacNAc (SLN) conjugates bind it with 103 -fold enhanced potency. Herein, we describe the oligomerization of distance-optimized bivalent binders by branched and linear hybridization on long repetitive DNA templates. The most effective complexes fully inhibited IAVs at a DNA template concentration of 10-9 m. Although a 10-2 m concentration of free trisaccharide ligand is required for full inhibition of the virus, DNA templating enables a 104 -fold reduction in the amount of sugar required. Notably, hybridization-induced rigidification of the DNA templates increased the serospecificity. Cryo-TEM analysis revealed that both spaghetti-type linear forms and cotton-ball-like clusters are able to bridge several adjacent HA molecules on the IAV surface. Programmed self-assembly of ligand-nucleic acid conjugates on long DNA templates might provide generic access to target-specific, high-affinity binders of proteins on globular objects such as cells and viruses.

Twinned Growth of Metal-Free, Triazine-Based Photocatalyst Films as Mixed-Dimensional (2D/3D) van der Waals Heterostructures.

Design and synthesis of ordered, metal-free layered materials is intrinsically difficult due to the limitations of vapor deposition processes that are used in their making. Mixed-dimensional (2D/3D) metal-free van der Waals (vdW) heterostructures based on triazine (C3 N3 ) linkers grow as large area, transparent yellow-orange membranes on copper surfaces from solution. The membranes have an indirect band gap (Eg,opt = 1.91 eV, Eg,elec = 1.84 eV) and are moderately porous (124 m2 g-1 ). The material consists of a crystalline 2D phase that is fully sp2 hybridized and provides structural stability, and an amorphous, porous phase with mixed sp2 -sp hybridization. Interestingly, this 2D/3D vdW heterostructure grows in a twinned mechanism from a one-pot reaction mixture: unprecedented for metal-free frameworks and a direct consequence of on-catalyst synthesis. Thanks to the efficient type I heterojunction, electron transfer processes are fundamentally improved and hence, the material is capable of metal-free, light-induced hydrogen evolution from water without the need for a noble metal cocatalyst (34 µmol h-1 g-1 without Pt). The results highlight that twinned growth mechanisms are observed in the realm of "wet" chemistry, and that they can be used to fabricate otherwise challenging 2D/3D vdW heterostructures with composite properties.