Massive assay for single viruses in single cells

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The genome of every eukaryotic cell is coated in a molecular meshwork called chromatin. As the first layer between genes and their environment, chromatin plays the roles of information repository and coordinator of gene expression. Importantly, genomes also contain genetic parasites such as transposons and integrated retroviruses. Through its ability to regulate gene expression, chromatin also acts as a safeguard of the genome against genetic aggressions. The molecular mechanisms by which chromatin defends the genome are still poorly understood. It has long been observed that transposons and retroviruses tend to be silenced by the host cell but it is still unknown how they are recognized and identified as threats. The aim of my research programme is to understand this particular mechanism of genetic immunity. More specifically, this project is designed to study how genetic aggressors are identified and silenced by the host cell. To study this important process, we must first develop a new genetic technology and bespoke software packages that will allow us to simultaneously track the fate of viruses in millions of individual cells. We are developing a novel high-throughput technology called SHIVA (Secondary Hyper-Indexing of Virus Activity) precisely for that purpose. With a complete record of parasite activity in every cell, we can identify patterns of expression that in turn inform us about the mechanism at work. With this approach, we can test the existence of recognition mechanisms to identify genetic parasites. We propose to establish a proof of principle using HIV as a model virus because of the large amount of available tools in this system. Importantly, the SHIVA technology can be used with any natural or artificial insertion sequences, including viruses, transposons and transgenes. Finally, we can combine SHIVA with genetic tools such as CRISPR screens to identify the genes that can recognize genetic parasites and contribute to the natural genetic immunity of eukaryotic cells. In summary, this research program paves the way to understand how eukaryotic genomes identify and neutralize integrated foreign genetic elements.