Purification and functional characterization of a biologically active full-length feline immunodeficiency virus (FIV) pr50^gag

The feline immunodeficiency virus (FIV) full-length Pr50^Gag precursor is a key player in the assembly of new viral particles. It is also a critical component of the efficient selection and packaging of two copies of genomic RNA (gRNA) into the newly formed virus particles from a wide pool of cellular and spliced viral RNA. To understand the molecular mechanisms involved during FIV gRNA packaging, we expressed the His₆-tagged and untagged recombinant FIV Pr50^Gag protein both in eukaryotic and prokaryotic cells. The recombinant Pr50^Gag-His₆-tag fusion protein was purified from soluble fractions of prokaryotic cultures using immobilized metal affinity chromatography (IMAC). This purified protein was able to assemble in vitro into virus-like particles (VLPs), indicating that it preserved its ability to oligomerize/multimerize. Furthermore, VLPs formed in eukaryotic cells by the FIV full-length Pr50^Gag both in the presence and absence of His₆-tag could package FIV sub-genomic RNA to similar levels, suggesting that the biological activity of the recombinant full-length Pr50^Gag fusion protein was retained in the presence of His₆-tag at the carboxy terminus. Successful expression and purification of a biologically active, recombinant full-length Pr50^Gag-His₆-tag fusion protein will allow study of the intricate RNA-protein interactions involved during FIV gRNA encapsidation.