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Cell entry of most alphaherpesviruses is mediated by the binding of glycoprotein D (gD) to different cell surface receptors. Equine herpesvirus type 1 (EHV-1) and EHV-4 gDs interact with equine major histocompatibility complex I (MHC-I) to initiate entry into equine cells. We have characterized the gD-MHC-I interaction by solving the crystal structures of EHV-1 and EHV-4 gDs (gD1, gD4), performing protein-protein docking simulations, surface plasmon resonance (SPR) analysis, and biological assays. The structures of gD1 and gD4 revealed the existence of a common V-set immunoglobulin-like (IgV-like) core comparable to those of other gD homologs. Molecular modeling yielded plausible binding hypotheses and identified key residues (F213 and D261) that are important for virus binding. Altering the key residues resulted in impaired virus growth in cells, which highlights the important role of these residues in the gD-MHC-I interaction. Taken together, our results add to our understanding of the initial herpesvirus-cell interactions and will contribute to the targeted design of antiviral drugs and vaccine development.Author summaryEquine herpesvirus type 1 (EHV-1) and type 4 (EHV-4) are endemic in horses and cause great suffering as well as substantial economic losses to the equine industry. Current vaccines do not prevent infections and treatment is difficult. A prerequisite for vaccine and drug development is an in-depth understanding of the virus replication cycle, especially the virus entry process in order to block the infection at early stages. Entry of alphaherpesviruses into the host cell is mediated by a set of virus envelope glycoproteins including glycoprotein D (gD) that triggers the internalization of the virus particle. The structure of gD and the interaction with the entry receptor equine major histocompatibility complex class I (MHC-I) remains elusive. Here, we solved the crystal structures of gD1 and gD4 that allowed us to model virus-receptor interaction and to determine the key residues for virus entry. Alterations of these key residues impaired virus growth in cell culture. The overall structure of gD1 and gD4 shows classical features of other alphaherpesvirus gDs making it possible to gain further insights into human pathogens as well.

Exposure of phosphatidylserine (PS) in the outer leaflet of the plasma membrane is induced by infection with several members of the Alphaherpesvirinae subfamily. There is evidence that PS is used by the equine herpesvirus type 1 (EHV-1) during entry, but the exact role of PS and other phospholipids in the entry process remains unknown. Here, we investigated the interaction of differently charged phospholipids with virus particles and determined their influence on infection. Our data show that liposomes containing negatively charged PS or positively charged DOTAP [N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium)] inhibited EHV-1 infection, while neutral phosphatidylcholine (PC) had no effect. Inhibition of infection with PS was transient, decreased with time, and was dose dependent. Our findings indicate that both cationic and anionic phospholipids can interact with the virus and reduce infectivity, while acting through different mechanisms. Charged phospholipids were found to have antiviral effects and can may be used to inhibit EHV-1 infection.

The matrix protein M1 of the Influenza A virus (IAV) is supposed to mediate viral assembly and budding at the plasma membrane (PM) of infected cells. In order for a new viral particle to form, the PM lipid bilayer has to bend into a vesicle toward the extracellular side. Studies in cellular models have proposed that different viral proteins might be responsible for inducing membrane curvature in this context (including M1), but a clear consensus has not been reached. In the present study, we use a combination of fluorescence microscopy, cryogenic transmission electron microscopy (cryo-TEM), cryo-electron tomography (cryo-ET) and scanning fluorescence correlation spectroscopy (sFCS) to investigate M1-induced membrane deformation in biophysical models of the PM. Our results indicate that M1 is indeed able to cause membrane curvature in lipid bilayers containing negatively charged lipids, in the absence of other viral components. Furthermore, we prove that protein binding is not sufficient to induce membrane restructuring. Rather, it appears that stable M1–M1 interactions and multimer formation are required in order to alter the bilayer three-dimensional structure, through the formation of a protein scaffold. Finally, our results suggest that, in a physiological context, M1-induced membrane deformation might be modulated by the initial bilayer curvature and the lateral organization of membrane components (i.e. the presence of lipid domains).

The matrix protein M1 of the Influenza A virus is considered to mediate viral assembly and budding at the plasma membrane (PM) of infected cells. In order for a new viral particle to form, the PM lipid bilayer has to bend into a vesicle towards the extracellular side. Studies in cellular models have proposed that different viral proteins might be responsible for inducing membrane curvature in this context (including M1), but a clear consensus has not been reached. In this study, we use a combination of fluorescence microscopy, cryogenic transmission electron microscopy (cryo-TEM), cryo-electron tomography (cryo-ET) and scanning fluorescence correlation spectroscopy (sFCS) to investigate M1-induced membrane deformation in biophysical models of the PM. Our results indicate that M1 is indeed capable to cause membrane curvature in lipid bilayers containing negatively-charged lipids, in the absence of other viral components. Furthermore, we prove that simple protein binding is not sufficient to induce membrane restructuring. Rather, it appears that stable M1-M1 interactions and multimer formation are required in order to alter the bilayer three-dimensional structure, through the formation of a protein scaffold. Finally, our results suggest that, in a physiological context, M1-induced membrane deformation might be modulated by the initial bilayer curvature and the lateral organization of membrane components (i.e. the presence of lipid domains).

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.

Influenza A virus is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. One of the ten proteins encoded by the viral genome, the matrix protein M1, is abundantly produced in infected cells and plays a structural role in determining the morphology of the virus. During assembly of new viral particles, M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. The structure of M1 is only partially known. In particular, structural details of M1 interactions with the cellular plasma membrane as well as M1–protein interactions and multimerization have not been clarified, yet.In this work, we employed a set of complementary experimental and theoretical tools to tackle these issues. Using raster image correlation, surface plasmon resonance and circular dichroism spectroscopies, we quantified membrane association and oligomerization of full-length M1 and of different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region, residues 95-105). Furthermore, we report novel information on structural changes in M1 occurring upon binding to membranes. Our experimental results are corroborated by an all-atom model of the full-length M1 protein bound to a negatively charged lipid bilayer.