Christian Sieben

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.

Heterogeneity of AMPA receptor trafficking and molecular interactions revealed by superresolution analysis of live cell imaging

Simultaneous tracking of many thousands of individual particles in live cells is possible now with the advent of high-density superresolution imaging methods. We present an approach to extract local biophysical properties of cell-particle interaction from such newly acquired large collection of data. Because classical methods do not keep the spatial localization of individual trajectories, it is not possible to access localized biophysical parameters. In contrast, by combining the high-density superresolution imaging data with the present analysis, we determine the local properties of protein dynamics. We specifically focus on AMPA receptor (AMPAR) trafficking and estimate the strength of their molecular interaction at the subdiffraction level in hippocampal dendrites. These interactions correspond to attracting potential wells of large size, showing that the high density of AMPARs is generated by physical interactions with an ensemble of cooperative membrane surface binding sites, rather than molecular crowding or aggregation, which is the case for the membrane viral glycoprotein VSVG. We further show that AMPARs can either be pushed in or out of dendritic spines. Finally, we characterize the recurrent step of influenza trajectories. To conclude, the present analysis allows the identification of the molecular organization responsible for the heterogeneities of random trajectories in cells.

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.

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.

Influenza virus binds its host cell using multiple dynamic interactions

Influenza virus belongs to a wide range of enveloped viruses. The major spike protein hemagglutinin binds sialic acid residues of glycoproteins and glycolipids with dissociation constants in the millimolar range [Sauter NK, et al. (1992) Biochemistry 31:9609–9621], indicating a multivalent binding mode. Here, we characterized the attachment of influenza virus to host cell receptors using three independent approaches. Optical tweezers and atomic force microscopy-based single-molecule force spectroscopy revealed very low interaction forces. Further, the observation of sequential unbinding events strongly suggests a multivalent binding mode between virus and cell membrane. Molecular dynamics simulations reveal a variety of unbinding pathways that indicate a highly dynamic interaction between HA and its receptor, allowing rationalization of influenza virus–cell binding quantitatively at the molecular level.

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.

Super-resolution imaging of multiple cells by optimized flat-field epi-illumination

An epi-illumination system based on microlens arrays enables field-independent imaging of multiple cells with nanoscale resolution and large field of views. Biological processes are inherently multi-scale, and supramolecular complexes at the nanoscale determine changes at the cellular scale and beyond. Single-molecule localization microscopy (SMLM)1,2,3 techniques have been established as important tools for studying cellular features with resolutions of the order of around 10 nm. However, in their current form these modalities are limited by a highly constrained field of view (FOV) and field-dependent image resolution. Here, we develop a low-cost microlens array (MLA)-based epi-illumination system—flat illumination for field-independent imaging (FIFI)—that can efficiently and homogeneously perform simultaneous imaging of multiple cells with nanoscale resolution. The optical principle of FIFI, which is an extension of the Kohler integrator, is further elucidated and modelled with a new, free simulation package. We demonstrate FIFIs capabilities by imaging multiple COS-7 and bacteria cells in 100 × 100 μm2 SMLM images—more than quadrupling the size of a typical FOV and producing near-gigapixel-sized images of uniformly high quality.

Influenza A Matrix Protein M1 Multimerizes upon Binding to Lipid Membranes

The matrix protein M1 plays a pivotal role in the budding of influenza virus from the plasma membrane (PM) of infected cells. This protein interacts with viral genetic material and envelope proteins while binding to the inner leaflet of the PM. Its oligomerization is therefore closely connected to the assembly of viral components and the formation of new virions. Of interest, the molecular details of M1 interaction with lipids and other viral proteins are far from being understood, and it remains to be determined whether the multimerization of M1 is affected by its binding to the PM and interaction with its components. To clarify the connection between M1 oligomerization and binding to lipid membranes, we applied a combination of several quantitative microscopy approaches. First, we used number and brightness (N&B) microscopy to characterize protein multimerization upon interaction with the PM of living cells. Second, we used controlled biophysical models of the PM (i.e., supported bilayers) to delve into the details of M1-lipid and M1-M1 interactions by employing a combination of raster image correlation spectroscopy (RICS), fluorescence correlation spectroscopy (FCS), and atomic force microscopy (AFM). Our results show that M1 oligomer formation is strongly enhanced by membrane binding and does not necessarily require the presence of other viral proteins. Furthermore, we propose a specific model to explain M1 binding to the lipid bilayer and the formation of multimers.

TORC1 organized in inhibited domains (TOROIDs) regulate TORC1 activity

The target of rapamycin (TOR) is a eukaryotic serine/threonine protein kinase that functions in two distinct complexes, TORC1 and TORC2, to regulate growth and metabolism. GTPases, responding to signals generated by abiotic stressors, nutrients, and, in metazoans, growth factors, play an important but poorly understood role in TORC1 regulation. Here we report that, in budding yeast, glucose withdrawal (which leads to an acute loss of TORC1 kinase activity) triggers a similarly rapid Rag GTPase-dependent redistribution of TORC1 from being semi-uniform around the vacuolar membrane to a single, vacuole-associated cylindrical structure visible by super-resolution optical microscopy. Three-dimensional reconstructions of cryo-electron micrograph images of these purified cylinders demonstrate that TORC1 oligomerizes into a higher-level hollow helical assembly, which we name a TOROID (TORC1 organized in inhibited domain). Fitting of the recently described mammalian TORC1 structure into our helical map reveals that oligomerization leads to steric occlusion of the active site. Guided by the implications from our reconstruction, we present a TOR1 allele that prevents both TOROID formation and TORC1 inactivation in response to glucose withdrawal, demonstrating that oligomerization is necessary for TORC1 inactivation. Our results reveal a novel mechanism by which Rag GTPases regulate TORC1 activity and suggest that the reversible assembly and/or disassembly of higher-level structures may be an underappreciated mechanism for the regulation of protein kinases.

Alteration of protein levels during influenza virus H1N1 infection in host cells: a proteomic survey of host and virus reveals differential dynamics

We studied the dynamics of the proteome of influenza virus A/PR/8/34 (H1N1) infected Madin-Darby canine kidney cells up to 12 hours post infection by mass spectrometry based quantitative proteomics using the approach of stable isotope labeling by amino acids in cell culture (SILAC). We identified 1311 cell proteins and, apart from the proton channel M2, all major virus proteins. Based on their abundance two groups of virus proteins could be distinguished being in line with the function of the proteins in genesis and formation of new virions. Further, the data indicate a correlation between the amount of proteins synthesized and their previously determined copy number inside the viral particle. We employed bioinformatic approaches such as functional clustering, gene ontology, and pathway (KEGG) enrichment tests to uncover co-regulated cellular protein sets, assigned the individual subsets to their biological function, and determined their interrelation within the progression of viral infection. For the first time we are able to describe dynamic changes of the cellular and, of note, the viral proteome in a time dependent manner simultaneously. Through cluster analysis, time dependent patterns of protein abundances revealed highly dynamic up- and/or down-regulation processes. Taken together our study provides strong evidence that virus infection has a major impact on the cell status at the protein level.