Kai Ludwig

Inhibition of Influenza Virus Infection by Multivalent Sialic‐Acid‐Functionalized Gold Nanoparticles

An efficient synthesis of sialic-acid-terminated glycerol dendron to chemically functionalize 2 nm and 14 nm gold nanoparticles (AuNPs) is described. These nanoparticles are highly stable and show high activity towards the inhibition of influenza virus infection. As the binding of the viral fusion protein hemagglutinin to the host cell surface is mediated by sialic acid receptors, a multivalent interaction with sialic-acid-functionalized AuNPs is expected to competitively inhibit viral infection. Electron microscopy techniques and biochemical analysis show a high binding affinity of the 14 nm AuNPs to hemagglutinin on the virus surface and, less efficiently, to isolated hemagglutinin. The functionalized AuNPs are nontoxic to the cells under the conditions studied. This approach allows a new type of molecular-imaging activity-correlation and is of particular relevance for further application in alternative antiviral therapy.

Structure of influenza haemagglutinin at neutral and at fusogenic pH by electron cryo-microscopy

The three‐dimensional structures of the complete haemagglutinin (HA) of influenza virus A/Japan/305/57 (H2N2) in its native (neutral pH) and membrane fusion‐competent (low pH) form by electron cryo‐microscopy at a resolution of 10 Å and 14 Å, respectively, have been determined. In the fusion‐competent form the subunits remain closely associated preserving typical overall features of the trimeric ectodomain at neutral pH. Rearrangements of the tertiary structure in the distal and the stem parts are associated with the formation of a central cavity through the entire ectodomain. We suggest that the cavity is essential for relocation of the so‐called fusion sequence of HA towards the target membrane.

Inhibition of Influenza Virus Activity by Multivalent Glycoarchitectures with Matched Sizes

We describe the synthesis of a series of sialic acid‐conjugated, polyglycerol‐based nanoparticles with diameters in the range of 1–100 nm. Particle sizes were varied along with the degree of functionalization to match the corresponding virus size and receptor multiplicity in order to achieve maximum efficiency. To build up these architectures, we used biocompatible, hyperbranched polyglycerols as scaffolds and recently developed polyglycerol‐based nanogels, the sizes of which can be varied between 2–4 nm and 40–100 nm, respectively. We demonstrate here that such multivalent nanoparticles inhibit influenza A virus cell binding and fusion and consequently infectivity. The potential of multivalency is evident from larger particles showing very efficient inhibition of viral infection up to 80 %. Indeed, both the size of the nanoparticle and the amount of ligand density are important determinants of inhibition efficiency. The inhibitory activity of the tested polymeric nanoparticles drastically increased with size. Particles with similar dimensions to the virus (50–100 nm) are exceedingly effective. We also observed a saturation point in degree of surface functionalization (i.e. ligand density), above which inhibition was not significantly improved. Our study emphasizes the importance of matching particle sizes and ligand densities to mimic biological surfaces and improve interactions; this is a vital concept underlying multivalent interactions.

Trapping of viruses in high-frequency electric field cages

High-frequency electric field cages can stably trap cells and microparticles in aqueous media [1, 2]. Such cages can be made by semiconductor fabricat ion techniques and are not to be confused with electromagnetic field devices used for t rapping atomic and elementary particles [3]. The behavior of various dielectric microparticles in uniform and nonuniform a.c. electric fields was investigated by Pohl in the 1970s and discussed in his monograph [4]. The mot ion of individual cells in nonuniform a.c. fields, termed dielectrophoresis (DP), was studied in subsequent decades [5]. The force, F, acting on a spherical dielectric particle of radius, r, in a timeperiodic electric field, f , can be expressed as a dipole approximat ion by

A New Family of Nonionic Dendritic Amphiphiles Displaying Unexpected Packing Parameters in Micellar Assemblies

In this paper we report on the synthesis of a new family of nonionic dendritic amphiphiles that self-assemble into defined supramolecular aggregates. Our approach is based on a modular architecture consisting of different generations of hydrophilic polyglycerol dendrons [G1-G3] connected to hydrophobic C(11) or C(16) alkyl chains via mono- or biaromatic spacers, respectively. All amphiphiles complex hydrophobic compounds as demonstrated by solubilization of Nile Red or pyrene. The structure of the supramolecular assemblies as well as the aggregation numbers are strongly influenced by the type of the dendritic headgroup. While the [G1] amphiphiles form different structures such as ringlike and fiberlike micelles, the [G2] and [G3] derivatives aggregate toward spherical micelles of low polydispersity clearly proven by transmission electron microscopy (TEM) measurements. In the case of the biaromatic [G2] derivative, the structural persistence of the micelles allowed a three-dimensional structure determination from the TEM data and confirmed the aggregation number obtained by static light scattering (SLS) measurements. On the basis of these data, molecular packing geometries indicate a drastic mass deficit of alkyl chains in the hydrophobic core volume of spherical micelles. It is noteworthy that these highly defined micelles contain as little as 15 molecules and possess up to 74% empty space. This behavior is unexpected as it is very different from classical detergent micelles such as sodium dodecyl sulfate (SDS), where the hydrophobic core volume is completely filled by alkyl chains.

Early steps of the conformational change of influenza virus hemagglutinin to a fusion active state: Stability and energetics of the hemagglutinin

A conformational change of the homotrimeric glycoprotein hemagglutinin (HA) of influenza virus mediates fusion between the viral envelope and the endosome membrane. The conformational change of the HA ectodomain is triggered by the acidic pH of the endosome lumen. An essential step of the conformational change is the formation of an extended coiled-coil motif exposing the hydrophobic fusion peptide toward the target membrane. The structures of the neutral-pH, non-fusion active conformation of the HA ectodomain and of a fragment of the ectodomain containing the coiled-coil motif are known. However, it is not known by which mechanism protonation triggers the conformational change of the stable neutral-pH conformation of the ectodomain. Here, recent studies on the stability of the HA ectodomain at neutral pH, the energetics of the conformational change toward the fusion-active state and of the unfolding of the HA ectodomain are summarised. A model for the early steps of the conformational change of the HA ectodomain is presented. The model implicates that protonation leads to a partial dissociation of the distal domains of the HA monomers that is driven by electrostatic repulsion. The opening of the ectodomain enables water to enter the ectodomain. The interaction of water with respective sequences originally shielded from contact with water drives the formation of the coiled-coil structure.

Receptor binding and pH stability — How influenza A virus hemagglutinin affects host-specific virus infection☆

Influenza A virus strains adopt different host specificities mainly depending on their hemagglutinin (HA) protein. Via HA, the virus binds sialic acid receptors of the host cell and, upon endocytic uptake, HA triggers fusion between the viral envelope bilayer and the endosomal membrane by a low pH-induced conformational change leading to the release of the viral genome into the host cell cytoplasm. Both functions are crucial for viral infection enabling the genesis of new progeny virus. Adaptation to different hosts in vitro was shown to require mutations within HA altering the receptor binding and/or fusion behavior of the respective virus strain. Human adapted influenza virus strains (H1N1, H3N2, H2N2) as well as recent avian influenza virus strains (H5, H7 and H9 subtypes) which gained the ability to infect humans mostly contained mutations in the receptor binding site (RBS) of HA enabling increased binding affinity of these viruses to human type (α-2,6 linked sialic acid) receptors. Thus, the receptor binding specificity seems to be the major requirement for successful adaptation to the human host; however, the RBS is not the only determinant of host specificity. Increased binding to a certain cell type does not always correlate with infection efficiency. Furthermore, viruses carrying mutations in the RBS often resulted in reduced viral fitness and were still unable to transmit between mammals. Recently, the pH stability of HA was reported to affect the transmissibility of influenza viruses. This review summarizes recent findings on the adaptation of influenza A viruses to the human host and related amino acid substitutions resulting in altered receptor binding specificity and/or modulated fusion pH of HA. Furthermore, the role of these properties (receptor specificity and pH stability of HA) for adaptation to and transmissibility in the human host is discussed. This article is part of a Special Issue entitled: Viral Membrane Proteins -- Channels for Cellular Networking.

The relevance of salt bridges for the stability of the influenza virus hemagglutinin

Hemagglutinin (HA) of influenza virus undergoes an irreversible conformational change at acidic pH, mediating viral fusion with the host endosomal membrane. To unravel the molecular basis of the pH‐dependent stability of HA, we demonstrate by mu‐tagenesis of the prototype HA of virus strain X31 (H3 subtype) that salt bridges, especially a tetrad salt bridge within the monomers, are crucial for folding and stability of the trimeric ectodomain. This complex (tetrad) salt bridge is highly conserved among influenza virus subtypes. Introducing additional sites of electrostatic attraction between monomers in the distal region enhanced the stability of ectodomain at low pH mimicking the natural variant H2 subtype. We propose that distinct salt bridges in the distal domain may contribute to the enhanced stability of HA of natural virus vari‐ants. This hypothesis may provide clues to understanding adaptations of virus strains (for example, avian influenza viruses) in order to preserve stability of the protein in the host‐specific environment.—Rachakonda, P. S., Veit, M., Korte, T., Ludwig, K., Böttcher, C., Huang, Q., Schmidt, M. F. G., Herrmann, A. The relevance of salt bridges for the stability of the influenza virus hemagglutinin. FASEB J. 21, 995–1002 (2007)

High frequency electric fields for trapping of viruses

Combining dielectrophoretic and hydrodynamic forces in micro electrode structures allows enrichment and stable trapping of viruses in aqueous solutions. Fluorescently labelled Influenza and Sendai viruses were collected from solutions of 2*105 – 2*108 viruses/μl within a few seconds. In the central part of the trap a virus aggregate of about 2–9 μm in diameter was formed. This corresponds to a local enrichment of viruses up to a factor of about 1400.

pH-Controlled two-step uncoating of influenza virus.

Upon endocytosis in its cellular host, influenza A virus transits via early to late endosomes. To efficiently release its genome, the composite viral shell must undergo significant structural rearrangement, but the exact sequence of events leading to viral uncoating remains largely speculative. In addition, no change in viral structure has ever been identified at the level of early endosomes, raising a question about their role. We performed AFM indentation on single viruses in conjunction with cellular assays under conditions that mimicked gradual acidification from early to late endosomes. We found that the release of the influenza genome requires sequential exposure to the pH of both early and late endosomes, with each step corresponding to changes in the virus mechanical response. Step 1 (pH 7.5-6) involves a modification of both hemagglutinin and the viral lumen and is reversible, whereas Step 2 (pH <6.0) involves M1 dissociation and major hemagglutinin conformational changes and is irreversible. Bypassing the early-endosomal pH step or blocking the envelope proton channel M2 precludes proper genome release and efficient infection, illustrating the importance of viral lumen acidification during the early endosomal residence for influenza virus infection.