Structural basis of NINJ1-mediated plasma membrane rupture in cell death.

Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event1-7. Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-18 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death.

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.

Influenza A viruses use multivalent sialic acid clusters for cell binding and receptor activation.

Influenza A virus (IAV) binds its host cell using the major viral surface protein hemagglutinin (HA). HA recognizes sialic acid, a plasma membrane glycan that functions as the specific primary attachment factor (AF). Since sialic acid alone cannot fulfill a signaling function, the virus needs to activate downstream factors to trigger endocytic uptake. Recently, the epidermal growth factor receptor (EGFR), a member of the receptor-tyrosine kinase family, was shown to be activated by IAV and transmit cell entry signals. However, how IAV's binding to sialic acid leads to engagement and activation of EGFR remains largely unclear. We used multicolor super-resolution microscopy to study the lateral organization of both IAV's AFs and its functional receptor EGFR at the scale of the IAV particle. Intriguingly, quantitative cluster analysis revealed that AFs and EGFR are organized in partially overlapping submicrometer clusters in the plasma membrane of A549 cells. Within AF domains, the local AF concentration reaches on average 10-fold the background concentration and tends to increase towards the cluster center, thereby representing a multivalent virus-binding platform. Using our experimentally measured cluster characteristics, we simulated virus diffusion on a flat membrane. The results predict that the local AF concentration strongly influences the distinct mobility pattern of IAVs, in a manner consistent with live-cell single-virus tracking data. In contrast to AFs, EGFR resides in smaller clusters. Virus binding activates EGFR, but interestingly, this process occurs without a major lateral EGFR redistribution, indicating the activation of pre-formed clusters, which we show are long-lived. Taken together, our results provide a quantitative understanding of the initial steps of influenza virus infection. Co-clustering of AF and EGFR permit a cooperative effect of binding and signaling at specific platforms, thus linking their spatial organization to their functional role during virus-cell binding and receptor activation.

Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation.

Transcription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-κB (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an "enhanceosome," nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.

Nanoscale Pattern Extraction from Relative Positions of Sparse 3D Localizations.

Inferring the organization of fluorescently labeled nanosized structures from single molecule localization microscopy (SMLM) data, typically obscured by stochastic noise and background, remains challenging. To overcome this, we developed a method to extract high-resolution ordered features from SMLM data that requires only a low fraction of targets to be localized with high precision. First, experimentally measured localizations are analyzed to produce relative position distributions (RPDs). Next, model RPDs are constructed using hypotheses of how the molecule is organized. Finally, a statistical comparison is used to select the most likely model. This approach allows pattern recognition at sub-1% detection efficiencies for target molecules, in large and heterogeneous samples and in 2D and 3D data sets. As a proof-of-concept, we infer ultrastructure of Nup107 within the nuclear pore, DNA origami structures, and α-actinin-2 within the cardiomyocyte Z-disc and assess the quality of images of centrioles to improve the averaged single-particle reconstruction.

Multicolor single-particle reconstruction of protein complexes.

Single-particle reconstruction (SPR) from electron microscopy (EM) images is widely used in structural biology, but it lacks direct information on protein identity. To address this limitation, we developed a computational and analytical framework that reconstructs and coaligns multiple proteins from 2D super-resolution fluorescence images. To demonstrate our method, we generated multicolor 3D reconstructions of several proteins within the human centriole, which revealed their relative locations, dimensions and orientations.

RNAi-based small molecule repositioning reveals clinically approved urea-based kinase inhibitors as broadly active antivirals.

Influenza viruses (IVs) tend to rapidly develop resistance to virus-directed vaccines and common antivirals targeting pathogen determinants, but novel host-directed approaches might preclude resistance development. To identify the most promising cellular targets for a host-directed approach against influenza, we performed a comparative small interfering RNA (siRNA) loss-of-function screen of IV replication in A549 cells. Analysis of four different IV strains including a highly pathogenic avian H5N1 strain, an influenza B virus (IBV) and two human influenza A viruses (IAVs) revealed 133 genes required by all four IV strains. According to gene enrichment analyses, these strain-independent host genes were particularly enriched for nucleocytoplasmic trafficking. In addition, 360 strain-specific genes were identified with distinct patterns of usage for IAVs versus IBV and human versus avian IVs. The strain-independent host genes served to define 43 experimental and otherwise clinically approved drugs, targeting reportedly fourteen of the encoded host factors. Amongst the approved drugs, the urea-based kinase inhibitors (UBKIs) regorafenib and sorafenib exhibited a superior therapeutic window of high IV antiviral activity and low cytotoxicity. Both UBKIs appeared to block a cell signaling pathway involved in IV replication after internalization, yet prior to vRNP uncoating. Interestingly, both compounds were active also against unrelated viruses including cowpox virus (CPXV), hantavirus (HTV), herpes simplex virus 1 (HSV1) and vesicular stomatitis virus (VSV) and showed antiviral efficacy in human primary respiratory cells. An in vitro resistance development analysis for regorafenib failed to detect IV resistance development against this drug. Taken together, the otherwise clinically approved UBKIs regorafenib and sorafenib possess high and broad-spectrum antiviral activity along with substantial robustness against resistance development and thus constitute attractive host-directed drug candidates against a range of viral infections including influenza.

Inhibition of influenza virus activity by the bovine seminal plasma protein PDC-109.

A number of viruses causing sexually transmissible diseases are transmitted via mammalian seminal plasma. Several components of seminal plasma have been shown to influence those viruses and their physiological impact. To unravel whether components of seminal plasma could affect viruses transmitted via other pathways, it was investigated here whether the bovine seminal plasma protein PDC-109, belonging to the Fn-type 2 protein family, influences the activity of influenza A viruses, used as a model for enveloped viruses. We found that PDC-109 inhibits the fusion of influenza virus with human erythrocyte membranes and leads to a decreased viral infection in MDCK cells. In the presence of the head group of the phospholipid phosphatidylcholine, phosphorylcholine, the inhibitory effect of PDC-109 was attenuated. This indicates that the impact of the protein is mainly caused by its binding to viral and to erythrocyte membranes thereby interfering with virus-cell binding. Our study underlines that Fn-type 2 proteins have to be considered as new antiviral components present in mammalian seminal plasma.

Viral RNA Degradation and Diffusion Act as a Bottleneck for the Influenza A Virus Infection Efficiency.

After endocytic uptake, influenza viruses transit early endosomal compartments and eventually reach late endosomes. There, the viral glycoprotein hemagglutinin (HA) triggers fusion between endosomal and viral membrane, a critical step that leads to release of the viral segmented genome destined to reach the cell nucleus. Endosomal maturation is a complex process involving acidification of the endosomal lumen as well as endosome motility along microtubules. While the pH drop is clearly critical for the conformational change and membrane fusion activity of HA, the effect of intracellular transport dynamics on the progress of infection remains largely unclear. In this study, we developed a comprehensive mathematical model accounting for the first steps of influenza virus infection. We calibrated our model with experimental data and challenged its predictions using recombinant viruses with altered pH sensitivity of HA. We identified the time point of virus-endosome fusion and thereby the diffusion distance of the released viral genome to the nucleus as a critical bottleneck for efficient virus infection. Further, we concluded and supported experimentally that the viral RNA is subjected to cytosolic degradation strongly limiting the probability of a successful genome import into the nucleus.

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.

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.

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.

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.

An Improved Workflow for the Quantification of Orthohantavirus Infection Using Automated Imaging and Flow Cytometry.

Determination of the infectious titer is a central requirement when working with pathogenic viruses. The plaque or focus assay is a commonly used but labor- and time-consuming approach for determining the infectious titer of orthohantavirus samples. We have developed an optimized virus quantification approach that relies on the fluorescence-based detection of the orthohantavirus nucleocapsid protein (N) in infected cells with high sensitivity. We present the use of flow cytometry but highlight fluorescence microscopy in combination with automated data analysis as an attractive alternative to increase the information retrieved from an infection experiment. Additionally, we offer open-source software equipped with a user-friendly graphical interface, eliminating the necessity for advanced programming skills.

Advanced fluorescence microscopy in respiratory virus cell biology.

Respiratory viruses are a major public health burden across all age groups around the globe, and are associated with high morbidity and mortality rates. They can be transmitted by multiple routes, including physical contact or droplets and aerosols, resulting in efficient spreading within the human population. Investigations of the cell biology of virus replication are thus of utmost importance to gain a better understanding of virus-induced pathogenicity and the development of antiviral countermeasures. Light and fluorescence microscopy techniques have revolutionized investigations of the cell biology of virus infection by allowing the study of the localization and dynamics of viral or cellular components directly in infected cells. Advanced microscopy including high- and super-resolution microscopy techniques available today can visualize biological processes at the single-virus and even single-molecule level, thus opening a unique view on virus infection. We will highlight how fluorescence microscopy has supported investigations on virus cell biology by focusing on three major respiratory viruses: respiratory syncytial virus (RSV), Influenza A virus (IAV) and SARS-CoV-2. We will review our current knowledge of virus replication and highlight how fluorescence microscopy has helped to improve our state of understanding. We will start by introducing major imaging and labeling modalities and conclude the chapter with a perspective discussion on remaining challenges and potential opportunities.

Advances in fluorescence microscopy for orthohantavirus research.

Orthohantaviruses are important zoonotic pathogens responsible for a considerable disease burden globally. Partly due to our incomplete understanding of orthohantavirus replication, there is currently no effective antiviral treatment available. Recently, novel microscopy techniques and cutting-edge, automated image analysis algorithms have emerged, enabling to study cellular, subcellular and even molecular processes in unprecedented detail and depth. To date, fluorescence light microscopy allows us to visualize viral and cellular components and macromolecular complexes in live cells, which in turn enables the study of specific steps of the viral replication cycle such as particle entry or protein trafficking at high temporal and spatial resolution. In this review, we highlight how fluorescence microscopy has provided new insights and improved our understanding of orthohantavirus biology. We discuss technical challenges such as studying live infected cells, give alternatives with recombinant protein expression and highlight future opportunities, for example, the application of super-resolution microscopy techniques, which has shown great potential in studies of different cellular processes and viral pathogens.

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.

Nanoscale organization of the endogenous ASC speck.

The NLRP3 inflammasome is a central component of the innate immune system. Its activation leads to formation of the ASC speck, a supramolecular assembly of the inflammasome adaptor protein ASC. Different models, based on ASC overexpression, have been proposed for the structure of the ASC speck. Using dual-color 3D super-resolution imaging (dSTORM and DNA-PAINT), we visualized the ASC speck structure following NLRP3 inflammasome activation using endogenous ASC expression. A complete structure was only obtainable by labeling with both anti-ASC antibodies and nanobodies. The complex varies in diameter between ∼800 and 1000 nm, and is composed of a dense core with emerging filaments. Dual-color confocal fluorescence microscopy indicated that the ASC speck does not colocalize with the microtubule-organizing center at late time points after Nigericin stimulation. From super-resolution images of whole cells, the ASC specks were sorted into a pseudo-time sequence indicating that they become denser but not larger during formation.

Relevance of Host Cell Surface Glycan Structure for Cell Specificity of Influenza A Viruses.

Influenza A viruses (IAVs) initiate infection via binding of the viral hemagglutinin (HA) to sialylated glycans on host cells. HA's receptor specificity towards individual glycans is well studied and clearly critical for virus infection, but the contribution of the highly heterogeneous and complex glycocalyx to virus-cell adhesion remains elusive. Here, we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS), to compare influenza virus receptor specificity with virus binding to live cells. Unexpectedly, we found that HA's receptor binding preference does not necessarily reflect virus-cell specificity. We propose SVFS as a tool to elucidate the cell binding preference of IAVs, thereby including the complex environment of sialylated receptors within the plasma membrane of living cells.