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

The simian immunodeficiency virus (SIV) precursor polypeptide Pr55Gag 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 Pr55Gag by expressing its different components independently, studies using full-length SIV Pr55Gag have not been conducted, primarily due to the unavailability of purified and biologically active full-length SIV Pr55Gag. We successfully expressed soluble, full-length SIV Pr55Gag 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 Pr55Gag which should facilitate future studies to understand protein structure and RNA-protein interactions involved during SIV gRNA packaging.

Automated summary

This study successfully expressed soluble and full-length SIV Pr55Gag and purified it using affinity and gel filtration chromatography. The purified Pr55Gag could assemble into virus-like particles in vitro and efficiently package SIV genomic RNA. This research provides an important method for future studies on protein structure and RNA-protein interactions during SIV gRNA packaging.