Title: Analysis of rhinoviruses replication complexes to identify host cell targets for the development of antiviral drugs for the treatment of asthma exacerbations
Funder: Asthma UK

Abstract: Rhinovirus infections in patients with asthma are the most common trigger for asthma exacerbations. However, no vaccine or antiviral treatment is currently available. The goal of this project is to identify cellular proteins important for rhinoviruses replication, which could be targeted by antiviral drugs in order to treat/prevent rhinoviruses-induced asthma exacerbations. Rhinoviruses replicate on cellular membranes, which they extensively remodel. This is believed to provide a favourable environment for the replication by providing a structural scaffold for the replication machinery, concentrating viral and cellular factors, and hiding the double-stranded RNA replication intermediates from the host cell defences. The formation of these replication complexes is still poorly understood. Two cellular proteins have been shown to be recruited to the replication complexes and to be essential for viral replication: the guanine nucleotide exchange factor GBF1 and the phosphatidylinositol-4-kinase PI4KIIIβ. However, the molecular mechanisms involved in their recruitment and their function in viral replication are still ill-defined. Moreover, other cellular proteins likely participate in viral replication. The objectives of this project are therefore to: 1) identify novel cellular proteins present in the rhinoviruses replication complexes and evaluate their role in viral replication 2) understand how GBF1 and PI4KIIIβ are recruited to rhinoviruses replication sites and identify potential new host factors important for replication, downstream of GBF1 and PI4KIIIβ. This will be achieved by a proteomics approach based on a method to determine in vivo interactomes of virulence factors that I developed. The strength of this method is that it allows the identification of host cell targets in infection conditions, unlike frequently used in vitro approaches which suffer from high rates of false positives or missed interactions. This is particularly important for rhinovirus infection, as some protein interactions probably only occur in the particular context of replication complexes. The replication complexes will be purified from infected cells by affinity purification of viral components of the replication complexes, after tagging them within the viral genome. The host proteins present in these complexes will be identified by mass spectrometry and the most promising ones will be selected for functional analysis. This will include assessment of whether the identified interactions are direct, confirmation of the recruitment of the identified host proteins to the replication sites, and, importantly, evaluation of their role in viral replication and the molecular mechanisms involved. The host cell targets downstream of GBF1 and PI4KIIIβ will be identified by comparing the protein composition of the replication complexes in the presence or absence of GBF1 or PI4KIIIβ inhibitors. Alternatively, for GBF1, its specific interactome in infected cells will be determined by tagging it directly and analysing its binding partners by pull-down and mass spectrometry. These experiments will also facilitate the identification of the molecular mechanisms of recruitment of GBF1 and PI4KIIIβ to the replication sites. This study will identify host proteins which are important for rhinoviruses replication, some of which should represent novel drug targets for the treatment of asthma exacerbations.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: RESPIRATORY;INFECTION