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

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.

Role of Purine-Rich Regions in Mason-Pfizer Monkey Virus (MPMV) Genomic RNA Packaging and Propagation

“…Transfections of HEK 293T cells with the aforementioned plasmids were carried out using the calcium phosphate transfection method (Invitrogen, United States) according to the manufacturer’s protocol along with pSEAP2-Control vector. The pSEAP2-Control vector expresses secreted alkaline phosphatase (SEAP) and was used to normalize for transfection efficiency of the assay using Great EscAPe SEAP Chemiluminescence kit 2.0 (Clontech, United States), as described previously ( Chameettachal et al, 2018 ; Pitchai et al, 2018 ; Krishnan et al, 2019 ). 27 h post-transfection, the pseudotyped virus particles produced from HEK 293T cells were harvested and clarified of cellular debris using low-speed centrifugation, and a portion of it was used to infect HeLaT4 target cells in the presence of 1 μg/ml DEAE dextran, a polycation polymer, to enhance infection efficiency.…”

Purification and Functional Characterization of a Biologically Active Full-Length Feline Immunodeficiency Virus (FIV) Pr50Gag

“…PCR amplified products with His 6 -tag (amplicon size 1379 base pairs) and without His 6 -tag (amplicon size 1361 base pairs) were digested with Xho I and cloned into the Xho I sites of the pcDNA3-a eukaryotic expression vector to generate AD2 and AD3, respectively. For the efficient nuclear export of Gag mRNA, the MPMV constitutive transport element (CTE; [63]) was cloned downstream of the gag stop codon, as described previously [14,15]. Both AD2 and AD3 clones were confirmed by sequencing.…”

“…For bacterial protein expression, VP10 was transformed into the BL21(DE3) strain of E. coli and cultured in an LB medium containing kanamycin. Analytical preparations of full-length recombinant Pr50 Gag His 6 -tag fusion protein were prepared as described previously [14,15].…”

“…However, not much is known about how the Gag precursor polypeptide achieves this selective gRNA packaging, primarily due to the lack of availability of a purified full-length Gag precursor protein. We and others have tried to narrow that gap by purifying these full-length Gag proteins for three different retroviruses that show different assembly/maturation processes: human immunodeficiency virus type 1 (HIV-1; [9,13]), a type C lentivirus that assembles at the plasma membrane, and the Mason–Pfizer monkey virus (MPMV; [14]) and mouse mammary tumor virus (MMTV; [15]), retroviruses that assemble intracellularly [16].…”

“…However, its appropriateness for in vitro RNA binding assays, such as band-shift assays and footprinting experiments, has not been established. Therefore, in our attempt to identify Gag binding site(s) on FIV gRNA, as a first step, we sub-optimally over-expressed recombinant full-length Pr50 Gag -His 6 -tag protein in E. coli and purified large amounts from bacterial soluble fractions employing immobilized metal affinity chromatography (IMAC) and high-pressure liquid chromatography (HPLC), as has recently been described for HIV-1, MPMV, and MMTV [13,14,15,60,61]. The purified recombinant full-length Pr50 Gag -His 6 -tag protein showed an intrinsic ability to assemble into virus-like particles in eukaryotic cells.…”