Humanized rabbit CD4 and CCR5 receptor complex supports HIV-1 entry into rabbit cells

Rabbits are being explored as potential model species for HIV-1 since rabbit cells display only few replication barriers to this virus: 1- they are not permissive to virus entry, 2- rabbit TRIM5 blocks HIV-1 reverse transcription, and 3- a primary rabbit macrophage-specific barrier reduces virion infectivity by a yet unknown mechanism. In light of generating a transgenic rabbit model that supports HIV-1 infection, our goal was to investigate whether minimal modifications to rabbit CD4 and CCR5 were sufficient to overcome the first barrier and thus may enable HIV-1 entry into rabbit cells. Additionally, we examined whether these modifications altered the physiological functions of the receptors. Computational modeling and evolutionary analyses of rabbit CD4 and CCR5 protein sequences identified amino acid residues in both receptors that are predicted to prevent HIV-1 entry into rabbit cells. Based on these in silico analyses, we generated a set of seven human-rabbit CD4 and CCR5 chimeras via site-directed mutagenesis and compared them to the respective wild-type proteins. Flow cytometry and immunofluorescence experiments confirmed that all chimeras were localized at the cell surface. Furthermore, all CD4 chimeras were down-modulated by HIV-1 Nef and Vpu by more than 55% and 70%, respectively. Cell-cell fusion assays revealed that the combination of humanized rabbit CD4 with humanized rabbit CCR5 enabled fusion mediated by the envelope proteins (Env) of R5-tropic HIV-1 strains. In line with this, humanized rabbit CD4 and CCR5 supported virion fusion of HIV-1 YU-2 and 49.5 strains as efficiently as the human HIV entry receptor complex. In summary, computational modeling allowed us to identify restrictions to HIV-1 entry into rabbit cells that could be overcome by mutagenesis of CD4 and CCR5. Thus, our findings contribute to the development of a fully permissive transgenic rabbit model for HIV-1 infection.