ABSTRACT
Visualizing virus-host interactions in situ inside infected cells by electron cryo-tomography provides unperturbed snapshots of the infection process. Here we focus on the assembly and egress pathway of herpesviruses. Cells infected with herpes simplex virus 1 produce and release not only infective virions but also non-infectious light particles (L-particles). L-particles are devoid of viral capsids and genomes. In this study, we analysed L-particle assembly and egress pathways in cultured dissociated hippocampus neurones by electron cryo-tomography. Virion and L-particle formation occurred in close proximity, suggesting shared assembly and exit pathways. Clathrin-like coats were occasionally associated with L-particle and virion assembly sites. Further, we compared the three-dimensional ultrastructure of intracellular and extracellular L-particles and quantified their diameters and the abundance of inclusion bodies contained.
Subject(s)
Herpesvirus 1, Human/physiology , Hippocampus/virology , Neurons/virology , Viral Proteins/metabolism , Virion/physiology , Virus Assembly , Virus Release , Adaptor Proteins, Vesicular Transport/metabolism , Adaptor Proteins, Vesicular Transport/ultrastructure , Animals , Chlorocebus aethiops , Cryoelectron Microscopy , Electron Microscope Tomography , Herpesvirus 1, Human/chemistry , Herpesvirus 1, Human/ultrastructure , Hippocampus/cytology , Host-Pathogen Interactions , Humans , Inclusion Bodies/metabolism , Inclusion Bodies/ultrastructure , Molecular Mimicry , Neurons/cytology , Neurons/metabolism , Neurons/ultrastructure , Particle Size , Rats , Vero Cells , Viral Proteins/ultrastructure , Virion/chemistry , Virion/ultrastructure , Virus ReplicationABSTRACT
Multiple entry receptors can mediate infection of cells by herpes simplex virus (HSV), permitting alternative pathways for infection and disease. We investigated the roles of two known entry receptors, herpesvirus entry mediator (HVEM) and nectin-1, in infection of neurons in the CNS and the development of encephalitis. Wild-type, HVEM KO, nectin-1 KO, and HVEM/nectin-1 double KO mice were inoculated with HSV into the hippocampus. The mice were examined for development of encephalitis or were killed at various times after inoculation for immunohistological analyses of brain slices. Nectin-1 KO mice showed no signs of disease after intracranial inoculation, and no HSV antigens were detectable in the brain parenchyma. However, HSV antigens were detected in non-parenchymal cells lining the ventricles. In the double KO mice, there was also no disease and no detectable expression of viral antigens even in non-parenchymal cells, indicating that infection of these cells in the nectin-1 KO mice was dependent on the expression of HVEM. Wild-type and HVEM KO mice rapidly developed encephalitis, and the patterns of HSV replication in the brain were indistinguishable. Thus, expression of nectin-1 is necessary for HSV infection via the intracranial route and for encephalitis; HVEM is largely irrelevant. These results contrast with recent findings that (i) either HVEM or nectin-1 can permit HSV infection of the vaginal epithelium in mice and (ii) nectin-1 is not the sole receptor capable of enabling spread of HSV infection from the vaginal epithelium to the PNS and CNS.
Subject(s)
Cell Adhesion Molecules/physiology , Encephalitis, Herpes Simplex/virology , Herpesvirus 2, Human/pathogenicity , Receptors, Virus/physiology , Animals , Antigens, Viral/metabolism , Cell Adhesion Molecules/deficiency , Cell Adhesion Molecules/genetics , Encephalitis, Herpes Simplex/physiopathology , Female , Herpesvirus 2, Human/immunology , Herpesvirus 2, Human/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nectins , Neurons/virology , Receptors, Tumor Necrosis Factor, Member 14/deficiency , Receptors, Tumor Necrosis Factor, Member 14/genetics , Receptors, Tumor Necrosis Factor, Member 14/physiology , Receptors, Virus/deficiency , Receptors, Virus/genetics , Virus InternalizationABSTRACT
The concerted action of four viral glycoproteins and at least one cellular receptor is required to catalyze herpes simplex virus 1 entry into host cells either by fusion at the plasma membrane or intracellularly after internalization by endocytosis. Here, we applied cryo electron tomography to capture 3D intermediates from Herpes simplex virus 1 fusion at the plasma membrane in their native environment by using two model systems: adherent cells and synaptosomes. The fusion process was delineated as a series of structurally different steps. The incoming capsid separated from the tegument and was closely surrounded by the cortical cytoskeleton. After entry, the viral membrane curvature changed concomitantly with a reorganization of the envelope glycoprotein spikes. Individual glycoprotein complexes in transitional conformations during pore formation and dilation revealed the complex viral fusion mechanism in action. Snapshots of the fusion intermediates provide unprecedented details concerning the overall structural changes occurring during herpesvirus entry. Moreover, our data suggest that there are two functional "poles" of the asymmetric herpesvirion: one related to cell entry, and the other formed during virus assembly.
Subject(s)
Cell Membrane/virology , Herpesvirus 1, Human/metabolism , Membrane Fusion , Animals , Capsid/metabolism , Cell Membrane/metabolism , Chlorocebus aethiops , Cryoelectron Microscopy/methods , Glycoproteins/chemistry , Glycoproteins/metabolism , Herpesviridae/metabolism , Male , Molecular Conformation , Potoroidae , Rats , Rats, Wistar , Synaptosomes/metabolism , Vero CellsABSTRACT
Glycoprotein B (gB) is a key component of the complex herpesvirus fusion machinery. We studied membrane interaction of two gB ectodomain forms and present an electron cryotomography structure of the gB-bilayer complex. The two forms differed in presence or absence of the membrane proximal region (MPR) but showed an overall similar trimeric shape. The presence of the MPR impeded interaction with liposomes. In contrast, the MPR-lacking form interacted efficiently with liposomes. Lateral interaction resulted in coat formation on the membranes. The structure revealed that interaction of gB with membranes was mediated by the fusion loops and limited to the outer membrane leaflet. The observed intrinsic propensity of gB to cluster on membranes indicates an additional role of gB in driving the fusion process forward beyond the transient fusion pore opening and subsequently leading to fusion pore expansion.
Subject(s)
Herpesvirus 1, Human/ultrastructure , Viral Envelope Proteins/chemistry , Cells, Cultured , Cryoelectron Microscopy , Crystallography, X-Ray , Humans , Lipid Bilayers/chemistry , Liposomes/chemistry , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Quaternary , Virus AttachmentABSTRACT
Caenorhabditis elegans proteins AFF-1 and EFF-1 [C. elegans fusion family (CeFF) proteins] are essential for developmental cell-to-cell fusion and can merge insect cells. To study the structure and function of AFF-1, we constructed vesicular stomatitis virus (VSV) displaying AFF-1 on the viral envelope, substituting the native fusogen VSV glycoprotein. Electron microscopy and tomography revealed that AFF-1 formed distinct supercomplexes resembling pentameric and hexameric "flowers" on pseudoviruses. Viruses carrying AFF-1 infected mammalian cells only when CeFFs were on the target cell surface. Furthermore, we identified fusion family (FF) proteins within and beyond nematodes, and divergent members from the human parasitic nematode Trichinella spiralis and the chordate Branchiostoma floridae could also fuse mammalian cells. Thus, FF proteins are part of an ancient family of cellular fusogens that can promote fusion when expressed on a viral particle.