Abstract
The mouse mammary tumor virus (MMTV) Pr77Gag polypeptide is an essential retroviral structural protein without which infectious viral particles cannot be formed. This process requires specific recognition and packaging of dimerized genomic RNA (gRNA) by Gag during virus assembly. Most of the previous work on retroviral assembly has used either the nucleocapsid portion of Gag, or other truncated Gag derivatives—not the natural substrate for virus assembly. In order to understand the molecular mechanism of MMTV gRNA packaging process, we expressed and purified full-length recombinant Pr77Gag-His6-tag fusion protein from soluble fractions of bacterial cultures. We show that the purified Pr77Gag-His6-tag protein retained the ability to assemble virus-like particles (VLPs) in vitro with morphologically similar immature intracellular particles. The recombinant proteins (with and without His6-tag) could both be expressed in prokaryotic and eukaryotic cells and had the ability to form VLPs in vivo. Most importantly, the recombinant Pr77Gag-His6-tag fusion proteins capable of making VLPs in eukaryotic cells were competent for packaging sub-genomic MMTV RNAs. The successful expression and purification of a biologically active, full-length MMTV Pr77Gag should lay down the foundation towards performing RNA–protein interaction(s), especially for structure-function studies and towards understanding molecular intricacies during MMTV gRNA packaging and assembly processes.
Highlights
The mouse mammary tumor virus (MMTV) is an oncogenic retrovirus that causes both breast cancer and lymphoma/leukemia in mice
The purified recombinant Pr77Gag -His6 -tag protein expressed in bacteria was resuspended in assembly buffer (50 mM Tris, 1.0 M NaCl) at a concentration of 2 mg/mL and incubated with yeast tRNA at a nucleic acid to protein ratio of 4% (w/w)
Mason-Pfizer monkey virus such as low temperature (28 C) and shorter duration (4 h only) as described in Materials and Methods. This was based on the earlier observations that in case of Mason-Pfizer monkey virus (MPMV) Pr78Gag, culturing bacteria at 37 ◦ C post-induction resulted in the confinement of MPMV Gag polyprotein in the inclusion bodies containing aberrantly assembled spiral like structures [70]
Summary
The mouse mammary tumor virus (MMTV) is an oncogenic retrovirus that causes both breast cancer and lymphoma/leukemia in mice. The Pr77Gag polyprotein plays a key role in selectively packaging the full length unspliced gRNA from a pool of cellular and spliced RNAs during viral assembly. Efficient expression and purification of full-length Pr77Gag should allow us to investigate the differential binding ability of this protein to the unspliced full-length gRNA during selective RNA packaging process. This should widen our understanding of the mechanisms of RNA–protein interaction(s) involved in gRNA packaging during MMTV life cycle
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