TRANSLATIONAL CONTROL OF VIRUSES BY ISG20 – TRAC-ISG20

-live imaging
-proteomic
-ribosome profiling
-eCLIP
-in vitro evolution
-characterization of retrieved mutants in vitro
-and in vivo

Partner 1 has performed RNAseq analyses on cells expressing or not ISG20 and are validating RNAs modulated by ISG20. We have constructed a functional ISG20-mcherry fusion protein that allow us to monitor dynamically the movement of ISG20 within the cell in various conditions. The data we have gathered indicates that ISG20 relocalizes to nuclear bodies we are currently characterizing during infection. This is the first observation of the dynamics of ISG20 and a strong link to its antiviral functions.

Partner#2 performed mass spectrometry analysis of ISG20 purified complexes under native and crosslinked conditions. The obtained results indicate a labile interaction of ISG20 with components of the mRNA translation machinery thus validating its role as a cellular factor involved in modulating mRNA translation. In parallel, partner 2 has set up the conditions to express and immuno-purify ISG20 in A549 cells infected with VSV in order to perform CLIP-seq and ribosome profiling experiments that are currently in preparation.

Partner 1 has so far been unable to obtain viral revertants of VSV resistant to ISG20 ex vivo. We are continuing this effort.

The next experiments will be carried out according to plan.

The results obtained so far point to key directions to decorticate the mechanism of action of ISG20. We are indeed confirming the decrease in specific RNAs regulated by ISG20 and will using different approaches described above continue to define the panorama of protein partners and RNAs specifically targeted by this protein.

Our ongoing studies have been presented to the Journe´es Francophones de Virologie, Strasbourg, France, 11-12/04/2022 and has been the subject of one review both with the PhD student as first author.

The interferon-sensitive gene 20 (ISG20) is a broad viral inhibitor previously thought to directly degrade invading viral RNA thanks to its RNase activity. This mechanism remained however controversial. In a recently published study, we have instead determined that ISG20 inhibits viral replication by impairing translation from viral RNAs and not by degrading them. Importantly, we also show that ISG20-mediated translation inhibition is distinct from previously known translation blocks (PKR, IFITs) and that ISG20 discriminates between mRNAs derived from chromosomal genes (self) and those originated from foreign genetic elements, be them viruses with a purely cytoplasmic life cycle or plasmid DNAs transcribed in the nucleus. Using orthogonal approaches, we want to identify the mechanism/s and specificities of ISG20 inhibition. In light of the current context, the characterization of the mechanism of translation inhibition by ISG20 will first focus on two RNA viruses: VSV as a model of negative-strand RNA viruses and SARS-CoV2 as a model for positive-strand RNA virus. The former will be used to decorticate and zoom into the mechanism of viral specificity and action of ISG20 in BSL2 conditions and the latter will be used to more finely characterize the specificity or differences with respect to viruses of high relevance for human health. After, the results obtained here will be extended to other viral pathogens as Zika or Retroviruses.
By studying a mechanism that broadly affects viral replication, we aim at characterizing a vulnerable step common to different viruses.