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Characterization of a novel mechanism restricting virus infection in reproductive tissues

Periodic Reporting for period 1 - RESTRIVIR (Characterization of a novel mechanism restricting virus infection in reproductive tissues)

Reporting period: 2016-04-01 to 2018-03-31

Viral infections lead every year to significant losses of human lives, livestock and plant crops, and are responsible for life-threatening pandemics and emerging epidemics, such as infuenza, HIV, ebola or MERS (Middle East Respiratory Syndrome). Viruses are obligatory intracellular pathogens, with a simple RNA- or DNA-based genome, sheltered in a small protein capsid, and hijack the cellular machinery for all stages of their life-cycle.
As in other insects and invertebrates, in the model organism Drosophila melanogaster, the main antiviral defence is the small interference (si)RNA pathway. In addition to RNAi, other pathways also participate in restricting viral infections in invertebrates, but their role and relative importance in complementing the RNAi response is not completely characterized
Previously, the host laboratory identified a putative novel antiviral restriction mechanism in the follicular somatic cells (FC) of the Drosophila ovarioles. The FC’s constitute an epithelial layer that encapsulates the germline and gives rise to the egg shell. Importantly, these are the last layer of somatic tissue blocking transmission of pathogens to the germline, preventing vertical transmission of viruses. The existence of an additional antiviral pathway in this tissue would then provide an extra layer of protection against virus infection for the next generations. With this project, we set out to identify the genes involved in this new pathway, using a combination of unbiased and targeted genetic screens, genomics and cell culture based assays. The combination of these approached allowed us to discard the involvement of additional RNAi pathways in this novel antiviral mechanism, and led to the identification of seven candidate loci, which are undergoing functional testing.
We performed an unbiased reverse genetic screen in the FCs of D. melanogaster, using a transgenic viral replicon system, tagged with GFP. Taking advantage of the Drosophila genetic toolbox, we carried out a deficiency mapping screen to determine loci genes involved in this pathway. We obtained seven candidate loci, and are in the process of validating candidate genes in these loci as being involved in the control of the replicon.
We also compared the gene expression and small RNA profiles of replicon restrictive and permissive FCs, to identify functional differences between these populations and obtain a list of candidates to test in an informed reverse genetic screen approach. The analysis of the small RNA profiles indicated that the siRNA pathway is the only active small RNA pathway against the replicon. The comparison of the transcriptomes of restrictive and permissive FCs led to the identification of 31 differentially expressed genes. However, knock down of the candidate genes in vivo did not affect replicon expression.
Finally, we established a cell culture model for this system. We stably transformed a FC derived cell line (OSS cells) with the GFP-tagged viral replicon. This ex vivo system was used to test the candidate genes, in parallel with the in vivo model. Again, we observed no differences in replicon expression after knock-down of the candidates.
During this period, I have published one review paper on the uses of the Drosophila model to discover novel antiviral factors. The results of this project were presented in two international conferences: European Drosophila Research Congress (2017, London) and European Congress of Entomology (2018, Napoli), in one joint laboratory meeting between the UPR9022 and the Vector Biology group of Elena Levashina (Max Planck Institute for Infection Biology, Berlin). The genetic model (transgenic flies expressing FHVΔB2-GFP replicon) was used in a teacher training action, co-organized between the host-laboratory and the Alsatian delegation of the “Maison pour la Science au Service des Professeurs” (MSA). I have trained one technician and one undergraduate student.
The goal of this project was to characterize a novel antiviral mechanism acting in the somatic epithelia that protects the Drosophila female germline. The conducted experiments – high throughput sequencing of small RNAs, sorting of sensitive/restrictive cells and characterization of their transcriptome, unbiased genetic screen and reconstitution of a tissue culture model to validate candidate genes ex vivo – led to the conclusion that i) the piRNA pathway is not involved in the control of the viral replicon; ii) the differentially expressed genes between restrictive/permissive cells do not appear to participate in the control of the replicon. These results suggest the involvement of a constitutive restriction mechanism, which remain to be identified. Characterization of the seven loci that I have identified is expected to provide hints to for the molecular mechanism at play in this reproductive tissue. This will also reveal whether evolutionarily conserved genes are involved, a finding that may have relevance for vertical transmission of viral pathogens in humans.