Purification and Functional Characterization of a Biologically Active Full-Length Feline Immunodeficiency Virus (FIV) Pr50.

The feline immunodeficiency virus (FIV) full-length Pr50 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 His-tagged and untagged recombinant FIV Pr50 protein both in eukaryotic and prokaryotic cells. The recombinant Pr50-His-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 both in the presence and absence of His-tag could package FIV sub-genomic RNA to similar levels, suggesting that the biological activity of the recombinant full-length Pr50 fusion protein was retained in the presence of His-tag at the carboxy terminus. Successful expression and purification of a biologically active, recombinant full-length Pr50-His-tag fusion protein will allow study of the intricate RNA-protein interactions involved during FIV gRNA encapsidation.