Abstract
MPMV precursor polypeptide Pr78Gag orchestrates assembly and packaging of genomic RNA (gRNA) into virus particles. Therefore, we have expressed recombinant full-length Pr78Gag either with or without His6-tag in bacterial as well as eukaryotic cultures and purified the recombinant protein from soluble fractions of the bacterial cultures. The recombinant Pr78Gag protein has the intrinsic ability to assemble in vitro to form virus like particles (VLPs). Consistent with this observation, the recombinant protein could form VLPs in both prokaryotes and eukaryotes. VLPs formed in eukaryotic cells by recombinant Pr78Gag with or without His6-tag can encapsidate MPMV transfer vector RNA, suggesting that the inclusion of the His6-tag to the full-length Pr78Gag did not interfere with its expression or biological function. This study demonstrates the expression and purification of a biologically active, recombinant Pr78Gag, which should pave the way to study RNA-protein interactions involved in the MPMV gRNA packaging process.
Highlights
Retroviruses are a group of viruses that require packaging/encapsidation of their “full-length”, unspliced, single-stranded, RNA genome into assembling viral particles for the continuity of their life cycle
The ψ sequences required for gRNA packaging have been identified as a structurally-conserved region generally present both upstream and downstream of the major splice donor within the 5′ untranslated region (5′ UTR) and often extending into the 5′ end of the Gag open reading frame (ORF)[1,2,3,4,5,6,8]
Among the proteins implicated in selective gRNA packaging into virus particles, the nucleocapsid (NC) region of the retroviral Gag polyprotein is a primary candidate, as this highly basic protein contains Cys-His boxes that can interact with Zn2+ ions to facilitate protein/RNA interactions[4,9]
Summary
Retroviruses are a group of viruses that require packaging/encapsidation of their “full-length”, unspliced, single-stranded, RNA genome (gRNA) into assembling viral particles for the continuity of their life cycle During this process, two copies of the gRNA dimerize and are preferentially packaged into the assembling virions compared to the spliced viral RNA and the large pool of cellular RNAs of the infected host cell[1,2,3,4,5,6,7]. It is thought that rather than recognizing monomeric RNA substrates, NC probably recognizes dimeric genomes, an interaction that is thought to initiate the multimerization of the Gag polyprotein on the RNA templates, eventually leading to encapsidation of the gRNA into the assembling virus particle[20,21,22] Together, these observations suggests that specific selection of gRNA from www.nature.com/scientificreports/. Cellular and spliced RNAs is a complex phenomenon that happens in the context of the whole Gag polyprotein, as has recently been shown for the human immunodeficiency virus type 1 (HIV-1)[23,24,25,26]
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