Project

Discipline

Morbillivirus; Non-cytolytic cell-to-cell spread; Brain and dermal cultures; Limited cell-cell fusion; Viral glycoproteins and cell surface receptor; Molecular mechansims; Persistent infection; pathogenesis; glycoproteins; recepotrs; microfusion

Lead
Lay summary
Morbilliviruses belong to the Paramyxovirinae subfamily and include a number of worldwide occurring highly virulent pathogens of which measles virus (MeV) in humans and the closely related canine distemper virus (CDV) in animals are the most important ones. The pathology of MeV and CDV infections is remarkably similar. Both infect the lymphoid tissues leading to severe immunosuppression, and a variety of epithelial tissues such as lung and skin. Both can induce a persistent infection of the brain leading to severe neurological disease, although neurological complications are more frequent in CDV than in MeV infection. Therefore, besides its severe impact on domestic and wild animal health, CDV infection in carnivores can be considered to be a translational model for MeV infection in humans.Initial viral interactions with specific target cells are events that shape the outcome of the disease. In Morbilliviruses these interactions are in part controlled by the envelope-anchored attachment protein (hemagglutinin, H), an essential viral component, which, assisted by the viral fusion (F) protein, initiates the viral entry process by binding a host cell-surface receptor. This proposal aims at identifying the molecular determinants of both CDV glycoproteins associated with restricted fusion activity induced by virulent CDV in glial and dermal cells. State-of-the-art functional and biochemical assays will be perform to map domains and identify residues of H and F that govern limited fusion activity. In parallel, cutting-edge techniques will be developed to identify new wild-type Morbillivirus cell entry molecules. Identification of new receptors combined with a comprehensive and detailed understanding of F/H-mediated cell-cell fusion will, in turn, set the stage for an accurate investigation of yet largely unexplored host-pathogens interactions sustaining persistent infection, the driving force triggering Morbillivirus-induced pathogenesis. Morbilliviruses remain a devastating group of pathogens: while MeV kills more than 100'000 people per year, CDV and rinderpest virus as well as Morbilliviruses of marine mammals continue to rage among an increasing range of terrestrial and aquatic animal species with high morbidity and mortality. Thus, infection of primary canine cells with highly pathogenic CDVs, for which an efficient reverse genetics technology is available, constitutes a unique translational model that will contribute to the understanding of the natural diseases induced by Morbilliviruses as a whole. Furthermore, this proposal will pave the way for the design of small molecules aiming at inhibiting viral cell-entry and prophylactic strategies.

Direct link to Lay Summary

Last update: 21.02.2013

Title	Year

Number	Title	Start	Funding scheme

Measles virus (MV) and canine distemper virus (CDV), both closely related Morbillivirus, can cause lethal brain infections. CDV induces a progressive neurological demyelinating disease in dogs resulting from the intrathecal antiviral immune response driven by viral persistence in the central nervous system (CNS). Our earlier work with virulent CDV in primary glial cells showed that viral persistence was associated with the ability of CDV to spread from cell-to-cell avoiding syncytia formation and subsequent cytolysis (as in infections with attenuated viruses) but still using the viral fusion machinery. The latter includes the viral fusion (F) protein, the viral hemagglutinin (H) protein and the host cell surface receptor interacting in such a way that cell-cell fusion is restricted to micro-pores allowing intercellular transfer of viral nucleocapsids.This proposal aims at identifying the molecular determinants of H and F associated with restricted fusion activity induced by virulent CDV in glial and dermal cells. In a first step, 3D homology models and transient expression systems we will be perform to map domains and identify residues of H and F that govern limited fusion activity. Cell lines engineered (or not) to express the CDV receptor CD150 (SLAM) will be used in combination with newly established cellular, biochemical and functional assays. While total protein expression will be determined by regular immunoblot, cell surface biotinylation will be undertaken to discriminate between intracellular and plasma membrane-exposed F and H antigenic materials. To strengthen these data, flow cytometry analysis using monoclonal antibodies (anti-F or H) will be used to monitor cell surface expression and potential modified conformational F and H structures. Additionally, to assess strength of F/H interaction, a recently established co-immunoprecipitation assay will be used. The latter procedure allows us to characterize the strength of interaction of H with functional F1 protein specifically at the plasma membrane. To determine H/receptor (SLAM)-binding activity, soluble forms of H and SLAM were produced and quantitative data will be recorded using a two colours-based flow cytometry system. Finally, fusion activity will be monitored by i) F/H-mediated syncytia formation assay, ii) reporter gene content mix assay and iii) bi-fluorescence-based F/H-induced pore formation assay. In a second step, specific mutations in F and H obtained in cell lines with the above mentioned assays will be dissected in the context of a complete viral infection. To this purpose, a reverse genetics system established from a highly virulent and demyelinating CDV strain (A75/17) in combination with either primary brain or dermal epithelial cells will be used. This technology already enabled us to engineer transgenic combined with specific glycoprotein-knockout recombinant viruses, which consequently were trans-complemented in lentivirus-transduced stable cell lines to enable initial cell-entry. This strategy recently offered us the unique opportunity to demonstrate a yet unappreciated role of the hemagglutinin protein in controlling non-cytolytic cell-to-cell CDV spread in primary glial cells. In addition to studies on the surface glycoproteins, our previous data provided strong evidence for the existence of yet unidentified CDV receptors both in astrocytes and epithelial footpad keratinocytes. Indeed, these cells have been shown to be deficient in SLAM expression, while supporting efficient wild-type CDV cell-to-cell spread. Consequently, this proposal additionally aims at identifying new CDV receptors. A cDNA library has already been generated from wild-type A75/17-CDV-susceptible primary dermal epithelial cells. The library contains approximately 5x105 independent and normalized clones, which will be cloned in a lentivirus-based expression system. Fluorescent protein-expressing recombinant wild-type CDVs will be used to screen a non-susceptible cell line, which will be priory transduced with the cDNA library-encoding lentivirus population. Fluorescent protein-expressing cells will be expanded and DNA inserts will be sequenced. The same strategy will be applied with a cDNA library generated from CNS cells of newborn dogs.Morbilliviruses infections continue to be a wide spread cause of diseases with high morbidity and mortality both in humans and animals. The CDV infection model presented in this proposal, combining primary SLAM-negative glial and dermal cells from the natural host with a virulent and demyelinating CDV strain for which a highly efficient reverse genetics technology is available, is unique and extremely relevant for the study of the molecular mechanisms of viral persistence in the CNS as well as in the skin. Viral persistence is a key factor in the establishment of detrimental diseases caused by these viruses. Thus, shedding light on the molecular mechanisms of persistence will potentially allow for the rational design of specific treatment of such diseases.