Cellular Membranes as a Playground for Semliki Forest Virus Replication Complex

All positive-strand RNA viruses utilize cellular membranes for the assembly of their replication complexes, which results in extensive membrane modification in infected host cells. These alterations act as structural and functional scaffolds for RNA replication, providing protection for the viral double-stranded RNA against host defences. It is known that different positive-strand RNA viruses alter different cellular membranes. However, the origin of the targeted membranes, the mechanisms that direct replication proteins to specific membranes and the steps in the formation of the membrane bound replication complex are not completely understood. Alphaviruses (including Semliki Forest virus, SFV), members of family Togaviridae, replicate their RNA in association with membranes derived from the endosomal and lysosomal compartment, inducing membrane invaginations called spherules. Spherule structures have been shown to be the specific sites for RNA synthesis. Four replication proteins, nsP1-nsP4, are translated as a polyprotein (P1234) which is processed autocatalytically and gives rise to a membrane-bound replication complex. Membrane binding is mediated via nsP1 which possesses an amphipathic α-helix (binding peptide) in the central region of the protein. The aim of this thesis was to characterize the association of the SFV replication complex with cellular membranes and the modification of the membranes during virus infection. Therefore, it was necessary to set up the system for determining which viral components are needed for inducing the spherules. In addition, the targeting of the replication complex, the formation site of the spherules and their intracellular trafficking were studied in detail. The results of current work demonstrate that mutations in the binding peptide region of nsP1 are lethal for virus replication and change the localization of the polyprotein precursor P123. The replication complex is first targeted to the plasma membrane where membrane invaginations, spherules, are induced. Using a specific regulated endocytosis event the spherules are internalized from the plasma membrane in neutral carrier vesicles and transported via an actin-and microtubule-dependent manner to the pericentriolar area. Homotypic fusions and fusions with pre-existing acidic organelles lead to the maturation of previously described cytopathic vacuoles with hundreds of spherules on their limiting membranes. This work provides new insights into the membrane binding mechanism of SFV replication complex and its role in the virus life cycle. Development of plasmid-driven system for studying the formation of the replication complex described in this thesis allows various applications to address different steps in SFV life cycle and virus-host interactions in the future. This trans-replication system could be applied for many different viruses. In addition, the current work brings up new aspects of membranes and cellular components involved in SFV replication leading to further understanding in the formation and dynamics of the membrane-associated replication complex.Positiivisäikeiset RNA virukset ovat kaikkein suurin virusryhmä, joka sisältää monia merkittäviä taudinaiheuttajia. Näiden virusten perimäaines on RNA:ta, joka voi suoraan soluun päästyään toimia lähetti-RNA:na. Kaikki positiivisäikeiset RNA virukset käyttävät hyväkseen solunsisäisiä kalvorakenteita, joiden yhteyteen ne rakentavat perimän monistamisesta vastaavat replikaatiokompleksinsa. Tämä johtaa infektoitujen solujen kalvorakenteiden laajamittaisiin muutoksiin. Kalvot toimivat virusreplikaation alustoina ja ne myös suojaavat monistamisen kaksisäikeistä RNA-välimuotoa solun puolustusmekanismeilta. Virusten käyttämien kalvojen alkuperä, se millä tavalla replikaatioproteiinit ohjautuvat kalvoille sekä replikaatikompleksin muodostumisen eri vaiheet ovat kuitenkin huonosti tunnettuja. Alfavirukset (mukaan lukien Semliki Forest virus, SFV) monistavat RNA:taan solun endosomien ja lysosomien kalvojen ulkopinnalla ja muodostavat näihin kuroumia, joita kutsutaan sferuleiksi. Juuri sferulit ovat RNA:n replikaation tapahtumapaikkoja. Virusten neljä replikaatioproteiinia (nsP1-nsP4) ohjautuvat kalvolle, joihin tarttumista välittää nsP1 proteiinin keskiosassa sijaitsevan amfifiilisen alfaheliksin välityksellä. Amfifiilisessä rakenteessa toinen puoli helikaalisesta spiraalista on hydrofobinen ja tarttuu kalvoon. Tämän väitöskirjan tavoitteena oli tarkastella, millä kalvomuutokset tapahtuvat SFV-infektion aikana soluissa. Väitöskirjassa osoitetaan, että mutaatiot amfifiilisessä heliksissä johtavat viruksen kyvyttömyyteen lisääntyä. Samat mutaatiot estävät myös replikaatioproteiinien tarttumisen kalvoihin. Toiseksi osoitetaan, että replikaatioproteiinit ohjautuvat ensin solukalvon sisäpinnalle, jossa sferulit muodostuvat. Käyttäen hyväkseen solun endosytoosikoneistoa ja solutukirangan aktiinia ja mikrotubuleita, sferulit siirtyvät kuljetusrakkuloissa tuman läheisyyteen. Siellä ne fuusioituvat solun happamiin endosomeihin ja lysosomeihin. Tämän tuloksena syntyy suuria kalvon ympäröimiä vakuoleja, joiden pinnalla on satoja sferuleita monistamassa viruksen perimäainesta. Kolmanneksi tässä työssä on rakennettu DNA-plasmidien transfektioon perustuva uusi menetelmä, jolla SFV:n replikaatiokompleksit voidaan rakentaa solussa erillisistä proteiini- ja RNA-komponenteista. Tällä menetelmällä tutkitaan replikaatiokompleksin muodostumisen vaiheita. Samanlaista menetelmää voidaan jatkossa soveltaa moniin eri viruksiin. Väitöskirjan perusteella voidaan paremmin ymmärtää viruksen replikaatiokompleksien syntyä ja liikkumista soluissa, mikä antaa jatkossa keinoja estää virusten lisääntymistä

VEGF-A/Notch-induced podosomes proteolyse basement membrane collagen-IV during retinal sprouting angiogenesis

During angiogenic sprouting, endothelial tip cells emerge from existing vessels in a process that requires vascular basement membrane degradation. Here, we show that F-actin/cortactin/P-Src-based matrix-degrading microdomains called podosomes contribute to this step. In vitro, VEGF-A/Notch signaling regulates the formation of functional podosomes in endothelial cells. Using a retinal neovascularization model, we demonstrate that tip cells assemble podosomes during physiological angiogenesis in vivo. In the retina, podosomes are also part of an interconnected network that surrounds large microvessels and impinges on the underlying basement membrane. Consistently, collagen-IV is scarce in podosome areas. Moreover, Notch inhibition exacerbates podosome formation and collagen-IV loss. We propose that the localized proteolytic action of podosomes on basement membrane collagen-IV facilitates endothelial cell sprouting and anastomosis within the developing vasculature. The identification of podosomes as key components of the sprouting machinery provides another opportunity to target angiogenesis therapeutically

Mutations at the palmitoylation site of non-structural protein nsP1 of Semliki Forest virus attenuate virus replication and cause accumulation of compensatory mutations

The replicase of Semliki Forest virus (SFV) consists of four non-structural proteins, designated nsP1–4, and is bound to cellular membranes via an amphipathic peptide and palmitoylated cysteine residues of nsP1. It was found that mutations preventing nsP1 palmitoylation also attenuated virus replication. The replacement of these cysteines by alanines, or their deletion, abolished virus viability, possibly due to disruption of interactions between nsP1 and nsP4, which is the catalytic subunit of the replicase. However, during a single infection cycle, the ability of the virus to replicate was restored due to accumulation of second-site mutations in nsP1. These mutations led to the restoration of nsP1–nsP4 interaction, but did not restore the palmitoylation of nsP1. The proteins with palmitoylation-site mutations, as well as those harbouring compensatory mutations in addition to palmitoylation-site mutations, were enzymically active and localized, at least in part, on the plasma membrane of transfected cells. Interestingly, deletion of 7 aa including the palmitoylation site of nsP1 had a relatively mild effect on virus viability and no significant impact on nsP1–nsP4 interaction. Similarly, the change of cysteine to alanine at the palmitoylation site of nsP1 of Sindbis virus had only a mild effect on virus replication. Taken together, these findings indicate that nsP1 palmitoylation as such is not the factor determining the ability to bind to cellular membranes and form a functional replicase complex. Instead, these abilities may be linked to the three-dimensional structure of nsP1 and the capability of nsP1 to interact with other components of the viral replicase complex

Nonlinear machine learning pattern recognition and bacteria-metabolite multilayer network analysis of perturbed gastric microbiome

The stomach is inhabited by diverse microbial communities, co-existing in a dynamic balance. Long-term use of drugs such as proton pump inhibitors (PPIs), or bacterial infection such as Helicobacter pylori, cause significant microbial alterations. Yet, studies revealing how the commensal bacteria re-organize, due to these perturbations of the gastric environment, are in early phase and rely principally on linear techniques for multivariate analysis. Here we disclose the importance of complementing linear dimensionality reduction techniques with nonlinear ones to unveil hidden patterns that remain unseen by linear embedding. Then, we prove the advantages to complete multivariate pattern analysis with differential network analysis, to reveal mechanisms of bacterial network re-organizations which emerge from perturbations induced by a medical treatment (PPIs) or an infectious state (H. pylori). Finally, we show how to build bacteria-metabolite multilayer networks that can deepen our understanding of the metabolite pathways significantly associated to the perturbed microbial communities

Variations sur le thème des podosomes, une affaire de contexte

Les podosomes sont des microdomaines membranaires riches en actine, en interaction directe avec la matrice extracellulaire. Des câbles d’acto-myosine les assemblent en réseau pour former une superstructure cellulaire aux fonctions versatiles. Extensivement décrits in vitro, les podosomes se dessinent comme des acteurs majeurs de processus physiologiques spécifiques. Les détails de leur intervention in vivo restent à préciser. Le microenvironnement ayant un effet prépondérant dans l’acquisition de leurs caractéristiques morphologiques et fonctionnelles, leur rôle ne peut être abordé que dans un contexte cellulaire particulier. Nous nous focaliserons ici sur trois processus impliquant ces structures et discuterons les propriétés des podosomes exploitées dans ces situations

Variations on the theme of podosomes: A matter of context

International audienceExtensive in vitro studies have described podosomes as actin-based structures at the plasma membrane, connecting the cell with its extracellular matrix and endowed with multiple capabilities. Contractile actin-myosin cables assemble them into a network that constitutes a multifaceted cellular superstructure taking different forms - with common characteristics - but manifesting different properties depending on the context of study. Their morphology and their role in cell functioning and behavior are therefore now apprehended in in vivo or in vitro situations relevant to physiological processes. We focus here on three of them, namely: macrophage migration, antigen presentation by dendritic cells and endothelial cell sprouting during angiogenesis to highlight the characteristics of podosomes and their functioning shaped by the microenvironment

Phosphatidylinositol 3-Kinase-, Actin-, and Microtubule-Dependent Transport of Semliki Forest Virus Replication Complexes from the Plasma Membrane to Modified Lysosomes▿ †

Like other positive-strand RNA viruses, alphaviruses replicate their genomes in association with modified intracellular membranes. Alphavirus replication sites consist of numerous bulb-shaped membrane invaginations (spherules), which contain the double-stranded replication intermediates. Time course studies with Semliki Forest virus (SFV)-infected cells were combined with live-cell imaging and electron microscopy to reveal that the replication complex spherules of SFV undergo an unprecedented large-scale movement between cellular compartments. The spherules first accumulated at the plasma membrane and were then internalized using an endocytic process that required a functional actin-myosin network, as shown by blebbistatin treatment. Wortmannin and other inhibitors indicated that the internalization of spherules also required the activity of phosphatidylinositol 3-kinase. The spherules therefore represent an unusual type of endocytic cargo. After endocytosis, spherule-containing vesicles were highly dynamic and had a neutral pH. These primary carriers fused with acidic endosomes and moved long distances on microtubules, in a manner prevented by nocodazole. The result of the large-scale migration was the formation of a very stable compartment, where the spherules were accumulated on the outer surfaces of unusually large and static acidic vacuoles localized in the pericentriolar region. Our work highlights both fundamental similarities and important differences in the processes that lead to the modified membrane compartments in cells infected by distinct groups of positive-sense RNA viruses

Role of the Amphipathic Peptide of Semliki Forest Virus Replicase Protein nsP1 in Membrane Association and Virus Replication

Semliki Forest virus RNA replication takes place in association with specific cytoplasmic vacuoles, derived from the endosomal apparatus. Of the four virus-encoded replicase proteins, nsP1 serves as the membrane anchor of the replication complex. An amphipathic peptide segment, G(245)STLYTESRKLLRSWHLPSV(264), has been implicated in the membrane binding of nsP1. nsP1 variants with changes within the peptide were studied after protein expression and in the context of virus infection. Proteins with mutations R253E and W259A accumulated in the cytoplasm and were very poorly palmitoylated. The same mutations also drastically affected the localization of the precursor polyprotein P123, and they were lethal when introduced into the virus genome. Mutations R253A and L255A+L256A partially changed the localization of nsP1, and the respective viruses acquired compensatory changes. L255A+L256A only yielded virus encoding L255A+L256V, indicating the importance of a hydrophobic residue in the central 256 position. When fused to green fluorescent protein, the peptide was required in at least two tandem copies to effect a change in localization, but even then the fusion protein was associated with membranes in a nonspecific manner. Thus, the amphipathic peptide is a crucial element for the membrane association of nsP1 and the replication complex. It provides essential affinity for membranes, and other regions of nsP1 also appear to contribute to the localization of the protein

A Methodology for Concomitant Isolation of Intimal and Adventitial Endothelial Cells from the Human Thoracic Aorta.

Aortic diseases are diverse and involve a multiplicity of biological systems in the vascular wall. Aortic dissection, which is usually preceded by aortic aneurysm, is a leading cause of morbidity and mortality in modern societies. Although the endothelium is now known to play an important role in vascular diseases, its contribution to aneurysmal aortic lesions remains largely unknown. The aim of this study was to define a reliable methodology for the isolation of aortic intimal and adventitial endothelial cells in order to throw light on issues relevant to endothelial cell biology in aneurysmal diseases.We set up protocols to isolate endothelial cells from both the intima and the adventitia of human aneurysmal aortic vessel segments. Throughout the procedure, analysis of cell morphology and endothelial markers allowed us to select an endothelial fraction which after two rounds of expansion yielded a population of >90% pure endothelial cells. These cells have the features and functionalities of freshly isolated cells and can be used for biochemical studies. The technique was successfully used for aortic vessel segments of 20 patients and 3 healthy donors.This simple and highly reproducible method allows the simultaneous preparation of reasonably pure primary cultures of intimal and adventitial human endothelial cells, thus providing a reliable source for investigating their biology and involvement in both thoracic aneurysms and other aortic diseases

Regulation of podosome formation in aortic endothelial cells vessels by physiological extracellular cues

International audienceInvadosomes are specialised actin-based dynamic microdomains of the plasma membrane. Their occurrence has been associated with cell adhesion, matrix degrading and mechanosensory functions that make them crucial regulators of cell migration and invasion. Monocytic, cancer cell and Src-transformed cell invadosomes have been extensively described. Less well defined are the structures which form in other cell types, i.e., non-haematopoietic and non-transformed cells, exposed to specific stimuli. We herein describe the specificities of podosomes induced in aortic endothelial cells stimulated with TGFβ in vitro and in conditions that more closely resemble the in vivo situation. These podosomes display the typical architecture of monocytic podosomes. They organise into large rosette-shape superstructures where they exhibit collective dynamic behavior consisting in cycles of formation and regression. At the ultrastructural level, microfilament arrangements in individual podosomes were revealed. Oxygen levels and hemodynamic forces, which are key players in endothelial cell biology, both influence the process. In 3D environment, podosomes appear as globular structures along cellular extensions. A better characterization of endothelial podosomes has far-reaching implications in the understanding and, possibly, in the treatment of some vascular diseases