Petr Chlanda

Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Hepatitis C Virus Replication

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.

Structural analysis of the roles of influenza A virus membrane-associated proteins in assembly and morphology.

ABSTRACT The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like. IMPORTANCE Influenza A virus is a major respiratory pathogen. It assembles membrane-enveloped virus particles whose shapes vary from spherical to filamentous. Here we examine the roles of individual viral proteins in mediating virus assembly and determining virus shape. To do this, we used a range of electron microscopy techniques to obtain and compare two- and three-dimensional images of virus particles and virus-like particles during and after assembly. The virus-like particles were produced using different combinations of viral proteins. Among our results, we found that coexpression of one or both of the viral surface proteins (hemagglutinin and neuraminidase) with the viral membrane-associated proteins encoded by the M segment results in assembly and release of filamentous virus-like particles in a manner very similar to that of the budding and release of influenza virions. These data provide novel insights into the roles played by individual viral proteins in influenza A virus assembly.

Reorganization of the endosomal system in Salmonella-infected cells: the ultrastructure of Salmonella-induced tubular compartments.

During the intracellular life of Salmonella enterica, a unique membrane-bound compartment termed Salmonella-containing vacuole, or SCV, is formed. By means of translocated effector proteins, intracellular Salmonella also induce the formation of extensive, highly dynamic membrane tubules termed Salmonella-induced filaments or SIF. Here we report the first detailed ultrastructural analyses of the SCV and SIF by electron microscopy (EM), EM tomography and live cell correlative light and electron microscopy (CLEM). We found that a subset of SIF is composed of double membranes that enclose portions of host cell cytosol and cytoskeletal filaments within its inner lumen. Despite some morphological similarities, we found that the formation of SIF double membranes is independent from autophagy and requires the function of the effector proteins SseF and SseG. The lumen of SIF network is accessible to various types of endocytosed material and our CLEM analysis of double membrane SIF demonstrated that fluid phase markers accumulate only between the inner and outer membrane of these structures, a space continual with endosomal lumen. Our work reveals how manipulation of the endosomal membrane system by an intracellular pathogen results in a unique tubular membrane compartmentalization of the host cell, generating a shielded niche permissive for intracellular proliferation of Salmonella.

The hemifusion structure induced by Influenza virus haemagglutinin is determined by physical properties of the target membranes

Influenza A virus haemagglutinin conformational change drives the membrane fusion of viral and endosomal membranes at low pH. Membrane fusion proceeds through an intermediate called hemifusion1,2. For viral fusion, the hemifusion structures are not determined3. Here, influenza virus-like particles4 carrying wild-type haemagglutinin or haemagglutinin hemifusion mutant G1S5 and liposome mixtures were studied at low pH by Volta phase plate cryo-electron tomography, which improves the signal-to-noise ratio close to focus. We determined two distinct hemifusion structures: a hemifusion diaphragm and a novel structure termed a ‘lipidic junction’. Liposomes with lipidic junctions were ruptured with membrane edges stabilized by haemagglutinin. The rupture frequency and hemifusion diaphragm diameter were not affected by G1S mutation, but decreased when the cholesterol level in the liposomes was close to physiological concentrations. We propose that haemagglutinin induces a merger between the viral and target membranes by one of two independent pathways: a rupture–insertion pathway leading to the lipidic junction and a hemifusion-stalk pathway leading to a fusion pore. The latter is relevant under the conditions of influenza virus infection of cells. Cholesterol concentration functions as a pathway switch because of its negative spontaneous curvature in the target bilayer, as determined by continuum analysis.

Open membranes are the precursors for assembly of large DNA viruses

Nucleo cytoplasmic large DNA viruses (NCLDVs) are a group of double‐stranded DNA viruses that replicate their DNA partly or entirely in the cytoplasm in association with viral factories (VFs). They share about 50 genes suggesting that they are derived from a common ancestor. Using transmission electron microscopy (TEM) and electron tomography (ET) we showed that the NCLDV vaccinia virus (VACV) acquires its membrane from open membrane intermediates, derived from the ER. These open membranes contribute to the formation of a single open membrane of the immature virion, shaped into a sphere by the assembly of the viral scaffold protein on its convex side. We now compare VACV with the NCLDV Mimivirus by TEM and ET and show that the latter also acquires its membrane from open membrane intermediates that accumulate at the periphery of the cytoplasmic VF. In analogy to VACV this membrane is shaped by the assembly of a layer on the convexside of its membrane, likely representing the Mimivirus capsid protein. By quantitative ET we show for both viruses that the open membrane intermediates of assembly adopt an ‘open‐eight’ conformation with a characteristic diameter of 90 nm for Mimi‐ and 50 nm for VACV. We discuss these results with respect to the common ancestry of NCLDVs and propose a hypothesis on the possible origin of this unusual membrane biogenesis.

Poxvirus membrane biogenesis: rupture not disruption

Enveloped viruses acquire their membrane from the host by budding at, or wrapping by, cellular membranes. Transmission electron microscopy (TEM) images, however, suggested that the prototype member of the poxviridae, vaccinia virus (VACV), may create its membrane ‘de novo’ with free open ends exposed in the cytosol. Within the frame of the German‐wide priority programme we re‐addressed the biogenesis and origin of the VACV membrane using electron tomography (ET), cryo‐EM and lipid analysis of purified VACV using mass spectrometry (MS). This review discussed how our data led to a model of unconventional membrane biogenesis involving membrane rupture and the generation of a single open membrane from open membrane intermediates. Lipid analyses of purified virus by MS suggest an ER origin with a relatively low cholesterol content compared with whole cells, confirming published data. Unlike previous reports using thin‐layer chromatography, no depletion of phosphatidylethanolamine was detected. We did detect, however, an enrichment for phosphatidic acid, diacylglycerol and phosphatidylinositol in the virion. Our data are discussed in the light of other pathogens that may requirecellular membrane rupture during their intracellular life cycle.

Eukaryotic-Like Virus Budding in Archaea

ABSTRACT Similar to many eukaryotic viruses (and unlike bacteriophages), viruses infecting archaea are often encased in lipid-containing envelopes. However, the mechanisms of their morphogenesis and egress remain unexplored. Here, we used dual-axis electron tomography (ET) to characterize the morphogenesis of Sulfolobus spindle-shaped virus 1 (SSV1), the prototype of the family Fuselloviridae and representative of the most abundant archaea-specific group of viruses. Our results show that SSV1 assembly and egress are concomitant and occur at the cellular cytoplasmic membrane via a process highly reminiscent of the budding of enveloped viruses that infect eukaryotes. The viral nucleoprotein complexes are extruded in the form of previously unknown rod-shaped intermediate structures which have an envelope continuous with the host membrane. Further maturation into characteristic spindle-shaped virions takes place while virions remain attached to the cell surface. Our data also revealed the formation of constricted ring-like structures which resemble the budding necks observed prior to the ESCRT machinery-mediated membrane scission during egress of various enveloped viruses of eukaryotes. Collectively, we provide evidence that archaeal spindle-shaped viruses contain a lipid envelope acquired upon budding of the viral nucleoprotein complex through the host cytoplasmic membrane. The proposed model bears a clear resemblance to the egress strategy employed by enveloped eukaryotic viruses and raises important questions as to how the archaeal single-layered membrane composed of tetraether lipids can undergo scission. IMPORTANCE The replication of enveloped viruses has been extensively studied in eukaryotes but has remained unexplored for enveloped viruses infecting Archaea. Here, we provide a sequential view on the assembly and egress of SSV1, a prototypic archaeal virus. The observed process is highly similar to the budding of eukaryotic enveloped viruses, including human immunodeficiency virus, influenza virus, and Ebola virus. The present study is the first to characterize such a phenomenon in archeal cells, showing that membrane budding is not an exclusive feature of eukaryotic viruses. Our results provide significant insights into the biogenesis and architecture of unique, spindle-shaped virions that infect archaea. Furthermore, our findings open doors for future inquiries into (i) the evolution of the virus budding process, (ii) mechanistic details of virus-mediated membrane scission in Archaea, and (iii) elucidation of virus- and host-encoded molecular players responsible for archaeal membrane and surface remodeling. The replication of enveloped viruses has been extensively studied in eukaryotes but has remained unexplored for enveloped viruses infecting bacteria and archaea. Here, we provide a sequential view on the assembly and egress of SSV1, a prototypic archaeal virus. The observed process is highly similar to the budding of eukaryotic enveloped viruses, including human immunodeficiency virus, influenza virus, and Ebola virus. The present study is the first to characterize such a phenomenon in archaeal cells, showing that membrane budding is not an exclusive feature of eukaryotic viruses. Our results provide significant insights into the biogenesis and architecture of unique, spindle-shaped virions that infect archaea. Furthermore, our findings open doors for future inquiries into (i) the evolution of the virus budding process, (ii) mechanistic details of virus-mediated membrane scission in Archaea, and (iii) elucidation of virus- and host-encoded molecular players responsible for archaeal membrane and surface remodeling.

Cryo-electron Microscopy of Vitreous Sections

More than 30 years ago two groups independently reported the vitrification of pure water, which was until then regarded as impossible without a cryoprotectant [1, 2]. This opened the opportunity to cryo-electron microscopy (cryo-EM) to observe biological samples at nanometer scale, close to their native state. However, poor electron penetration through biological samples sets the limit for sample thickness to less than the average size of the mammalian cell. In order to image bulky specimens at the cell or tissue level in transmission electron microscopy (TEM), a sample has to be either thinned by focused ion beam or mechanically sectioned. The latter technique, Cryo-Electron Microscopy of Vitreous Section (CEMOVIS), employs cryo-ultramicrotomy to produce sections with thicknesses of 40-100 μm of vitreous biological material suitable for cryo-EM. CEMOVIS consists of trimming and sectioning a sample with a diamond knife, placing and attaching the section onto an electron microscopy grid, transferring the grid to the cryo-electron microscope and imaging. All steps must be carried on below devitrification temperature to obtain successful results. In this chapter we provide a step-by-step guide to produce and image vitreous sections of a biological sample.

Protein–lipid interactions critical to replication of the influenza A virus

Influenza A virus (IAV) assembles on the plasma membrane where viral proteins localize to form a bud encompassing the viral genome, which ultimately pinches off to give rise to newly formed infectious virions. Upon entry, the virus faces the opposite task—fusion with the endosomal membrane and disassembly to deliver the viral genome to the cytoplasm. There are at least four influenza proteins—hemagglutinin (HA), neuraminidase (NA), matrix 1 protein (M1), and the M2 ion channel—that are known to directly interact with the cellular membrane and modify membrane curvature in order to both assemble and disassemble membrane‐enveloped virions. Here, we summarize and discuss current knowledge of the interactions of lipids and membrane proteins involved in the IAV replication cycle.

Vaccinia virus lacking A17 induces complex membrane structures composed of open membrane sheets.

The vaccinia virus (VACV) precursor membrane, the crescent, consists of an open membrane sheet and is formed by rupture of a cellular compartment. Here, we asked whether A17, a viral membrane protein, plays a role in membrane rupture. Without A17 synthesis, crescents are not formed, and instead, tubular and vesicular membranes accumulate (Rodriguez et al. in J Virol 69:4640–4648, 1). We used electron tomography (ET) to analyze whether the viral membranes lacking A17 consist of open membrane sheets. Tubular, vesicular and so far not described onion-shaped membranes, which consisted of open membrane sheets, were seen. Thus, the data show that membrane rupture occurs independently of the A17 protein.