APOBEC1 OLIGOMERIZATION AND LOCALIZATION IN RNA EDITING AND LENTIVIRAL RESTRICTION

APOBEC1 oligomerization and localization in RNA editing and lentiviral restriction In the past years, the emerging field of epitranscriptomic regulation of the cell has reignited interest in APOBEC1, an RNA editing enzyme. APOBEC1 is physiologically part of the editosome complex that deaminates a CAA codon into UAA Stop codon in the mRNA of Apolipoprotein B (ApoB), a protein essential for the transport of lipids in the blood. Yet, ApoB is the only fully characterized target of APOBEC1 among hundreds of recently identified ones. In the years of my PhD I have thus investigated APOBEC1 from two different point of view: on one hand I have investigated the molecular properties that define APOBEC1 activity on RNA, on the other one I have looked into its potential as a viral restriction agent. Starting from the observation that catalytically inactive mutants of APOBEC1 can have a dominant negative effect on its activity, I analysed in live cells APOBEC1 activity using a fluorescent reporter and flow cytometry. By titrating of APOBEC1 mediated RNA editing using inactive APOBEC1 chimeras, I could demonstrate that APOBEC1 ability to dimerize, a common property of other paralogues of the gene family, is not necessary for its RNA editing activity. Furthermore, I showed that the presence of dimerization-proficient inactive APOBEC1 increases the levels of a chimera of APOBEC1 unable to dimerize, thus leading to an increased activity. This is likely due to the dimer competing for the proteasomal degradation. From another perspective, APOBEC1 is just one of the member of the AID/APOBECs, a group of proteins part of adaptive and innate immunity. Similarly to the APOBEC3s, APOBEC1 is able to restrict the infectivity of retro/lentiviruses and the mobility of retrotransposons. Nevertheless, APOBEC1s from different species display varying levels of efficiency: among them, the human homologue is the least active. Starting from the hypothesis that its cellular localisation could be a determinant of its antiviral activity, we have used rat and human APOBEC1s tagged with an EGFP at their amino terminus. Compared to the wild-type APOBEC1, these chimeras show increased cytoplasmic retention and ability to inhibit HIV. Moreover, deletion mutants of APOBEC1 lacking the C-terminal Nuclear Export Signal result inactive against HIV. They regain their activity after alteration of their intracellular localisation with the EGFP tag. These findings suggest that the availability of APOBEC1 in the cytoplasmic compartment dramatically increases its ability to restrict HIV infectivity. Considering that nucleus-cytoplasmic shuttling of APOBEC1 is a regulated process, under appropriate conditions (cell type and state) the presence of human APOBEC1 in the cytoplasm could be relevant in conferring resistance against lentiviral infections.