Towards A Mechanistic Understanding of Gammaretrovirus Recognition of Host Receptors

Feline leukemia virus (FeLV) enters host cells by exploiting multipass solute carriers as receptors: the heme exporter FLVCR1a (SLC49A1) for FeLV-C and the phosphate transporter PiT-1 (SLC20A1) for FeLV-B. It remains unclear whether the viral glycoprotein recognises a specific conformational state of these transporters, and whether cargo occupancy, by biasing the conformational equilibrium, modulates that recognition. No structure of FLVCR1a or PiT-1 in complex with a viral envelope protein has yet been reported. This thesis combined biophysical analyses, membrane-protein production, and antibody-based tool development to address these questions. Building on established protocols for the purification of human FLVCR1a, reported to bind to the FeLV-type C, I used microscale thermophoresis and nano- differential scanning fluorimetry to show that hemin binds purified human FLVCR1a and increases the affinity of the FeLV-C surface-unit receptor-binding domain for the receptor by approximately sixfold. To my knowledge, this is the first biophysical evidence that substrate occupancy can influence the activity of FLVCR1a as a retroviral receptor, consistent with a conformational model of regulation that remains to be tested directly. Size-exclusion chromatography supported complex formation under these conditions, although the chromatographic conditions require further optimisation before the complex can be isolated as a homogeneous species. In parallel, in order to improve the affinity between the FeLV-type B-SU and the receptor, the feline orthologue was expressed and purified. Two further strands of work were undertaken to enable future studies. To extend the approach beyond a single receptor–envelope pair, I produced the FeLV-B receptor-binding domain together with its receptor, PiT-1, establishing the groundwork for a comparative analysis of FeLV receptor engagement. To support structural work on FLVCR1a, a panel of 16 monoclonal antibodies against human FLVCR1a was characterised, and seven clones bound the native receptor. One of these, 339F1, appears to recognise a conformational epitope and is therefore a promising candidate as a fiducial marker for cryo-EM. In conclusion, these results link transporter-substrate occupancy to retroviral receptor function in vitro and provide reagents and a methodological basis for future structural studies of FeLV receptor engagement.