Biochemical and Functional Characterization of Mouse Mammary Tumor Virus Full-Length Pr77Gag Expressed in Prokaryotic and Eukaryotic Cells

• Published in: Viruses Vol. 10; no. 6; p. 334
• Main Authors: Chameettachal, Akhil, Pillai, Vineeta, Ali, Lizna, Pitchai, Fathima, Ardah, Mustafa, Mustafa, Farah, Marquet, Roland, Rizvi, Tahir
• Format: Journal Article
• Language: English
• Published: Basel MDPI AG 18.06.2018; MDPI
• Subjects: Bacteria; Binding sites; Biological activity; Breast cancer; E coli; Fusion protein; Gag protein; Gene expression; Genomes; gRNA; Health sciences; HIV; Human immunodeficiency virus; mouse mammary tumor virus (MMTV); Nucleocapsids; Packaging; Pr77Gag; protein assembly; protein expression; protein purification; Proteins; retrovirus; Ribonucleic acid; RNA; RNA packaging; RNA–Gag interactions; RNA–protein interaction; Structure-function relationships; Tumor viruses; Virus-like particles; Viruses; United States > US; United Arab Emirates
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v10060334

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.