Collaborative project with UMR Virologie (INRA, ANSES, ENVA), Maisons-Alfort, France.
Flaviviruses, which are principally transmitted by mosquitoes and ticks, cause devastating human diseases such as Dengue fever in the tropics and subtropics and are a constant source of emerging pathogens of pandemic potential (such as ZIKA and Usutu viruses). Relative to the gravity of flaviviral infections and the recognized threat of future outbreaks, current understanding of flaviviral pathogenesis – and more particularly as regards tick-borne flaviviruses – is disproportionately underdeveloped.
Like all viruses, flaviviruses are obligate intracellular life forms whose survival requires subversion of metabolic circuits and evasion of anti-viral pathways. Within the cell, these pathways are embedded in extensive protein-protein interaction (PPI) networks, and their misappropriation by viruses is largely mediated by binary interactions between dedicated viral proteins and critical network proteins. Indeed, the targeted host proteins tend to be highly connected, such that the functional impact of a single interaction may be transmitted well beyond its immediate neighbourhood. Virus-host PPI thus represent molecular determinants of critical pathobiologic traits of flaviviruses, including host-range, zoonotic potential and virulence. Such interactions represent realistic targets for anti-viral therapies, thus providing a compelling reason to resolve the complete set of virus-cell interactions at the molecular level.
Comparative analysis of PPI established by different viruses is emerging as a means of discerning those responsible for particular pathobiological traits. We have recently performed a high throughput screen for protein-protein interactions involving the entire set of open reading frames for two tick-borne flaviviruses of concern to human and veterinary health —the tick-borne encephalitis and louping ill viruses (TBEV and LIV), respectively — and cDNA libraries of human and ruminant hosts. We have established a large data set of shared and virus-specific PPI, most of which have never been documented in the literature.
In the course of the hIPsTER project, state-of-the-art wet lab and in silico approaches will be applied to elucidate the biological meaning of these virus-host PPIs in flaviviral pathogenesis. In particular, we intend to define the functional significance of these PPI in viral infection as enhancing or restricting factors using an RNA interference (RNAi) approach. We will then determine their role in susceptibility or resistance of neural cells to infection and in neuropathogenesis in a recently developed pathological model of TBEV infection (co-cultures of human neuronal/glial cells derived from fetal neural progenitors) that reproduces major hallmarks of natural infection, such as high neuronal tropism and neuronal death. For selected PPI, we will investigate the mechanisms by which they disarm critical anti-viral defense pathways, and more particularly the type 1 interferon system, whose suppression is a virtual sine qua non for successful viral infection. We will explore the impact of the PPIs on the most salient biological processes of the human PIN through in silico analyses using Graph theory, and finally, perturbation of the vicinal protein-protein interaction network by viral proteins will be directly addressed in wet lab experiments using affinity purification coupled with mass spectrometry. For the latter, targeted cellular proteins will be selected on the basis of both viral and topological criteria; that is, viral-specificity and/or high degree of network connectivity.
We expect this work to illuminate the strategies by which tick-borne flaviviruses control cellular processes and cause disease, and ultimately disclose viral vulnerabilities that can be exploited therapeutically.
Beyond its ramifications for tick-borne flaviviruses, we expect hIPsTER to serve as a new paradigm for network-oriented analyses of viral pathobiology.