Expression, purification, and functional characterization of soluble recombinant full-length simian immunodeficiency...

The simian immunodeficiency virus (SIV) precursor polypeptide Pr55^Gag drives viral assembly and facilitates specific recognition and packaging of the SIV genomic RNA (gRNA) into viral particles. While several studies have tried to elucidate the role of SIV Pr55^Gag by expressing its different components independently, studies using full-length SIV Pr55^Gag have not been conducted, primarily due to the unavailability of purified and biologically active full-length SIV Pr55^Gag. We successfully expressed soluble, full-length SIV Pr55^Gag with His₆-tag in bacteria and purified it using affinity and gel filtration chromatography. In the process, we identified within Gag, a second in-frame start codon downstream of a putative Shine-Dalgarno-like sequence resulting in an additional truncated form of Gag. Synonymously mutating this sequence allowed expression of full-length Gag in its native form. The purified Gag assembled into virus-like particles (VLPs) in vitro in the presence of nucleic acids, revealing its biological functionality. In vivo experiments also confirmed formation of functional VLPs, and quantitative reverse transcriptase PCR demonstrated efficient packaging of SIV gRNA by these VLPs. The methodology we employed ensured the availability of >95% pure, biologically active, full-length SIV Pr55^Gag which should facilitate future studies to understand protein structure and RNA-protein interactions involved during SIV gRNA packaging.