Expression, purification, and characterization of biologically active full-length Mason-Pfizer monkey virus (MPMV) Pr78Gag

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.

Stabilizing role of structural elements within the 5´ Untranslated Region (UTR) and gag sequences in Mason-Pfizer monkey virus (MPMV) genomic RNA packaging

“…After infection, target cells are selected with media containing hygromycin B, allowing only those cells to survive which have been successful infected since the packaged RNA contains the hygromycin resistance gene cassette. Part of the figure adapted from Pitchai et al [68]. Figure 3.…”

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.…”

“…The remaining virus supernatant was clarified of cellular debris by passing through a 0.22-μm cellulose acetate syringe filter and then ultracentrifuged at 70,000 × g for 2 h at 4°C on a 20% ( w/v ) sucrose cushion to concentrate the virus particles, as described previously ( Jaballah et al, 2010 ; Kalloush et al, 2016 , 2019 ; Pitchai et al, 2018 ). The pelleted virus particles were resuspended in TNE buffer [0.25 M Tris (pH 8.0), 0.1 M NaCl, 0.001 M EDTA] and stored in Trizol Reagent (Invitrogen, United States) for virion RNA isolation and subsequently used for real-time quantitative PCR (RT-qPCR).…”

“…The cytoplasmic and packaged viral RNAs were isolated from Trizol following manufacturer’s instructions (Invitrogen, United States). Two micrograms of cellular RNA and the entire virion RNA prep from a six-well plate were subjected to Dnase-treatment (Turbo Dnase, Ambion, United States), followed by PCR amplification with MPMV-vector specific primers OTR1161 and OTR1163 ( Supplementary Table 1 ) to confirm the lack of any carryover plasmid contamination, as described previously ( Kalloush et al, 2016 ; Pitchai et al, 2018 ). The samples were then converted into cDNAs using M-MLV reverse transcriptase (Promega, United States), and random hexamers, as described earlier ( Jaballah et al, 2010 ; Kalloush et al, 2016 ).…”

“…Mason-Pfizer monkey virus is a betaretrovirus that forms intracytoplasmic virus particles unlike other retroviruses, except mouse mammary tumor virus (MMTV; Coffin et al, 1997 ). Because of the possible use of MPMV-based vectors in human gene therapy, its replicative biology has been investigated extensively with major emphasis on gRNA dimerization, packaging, and RNA propagation ( Vile et al, 1992 ; Harrison et al, 1995 ; Guesdon et al, 2001 ; Schmidt et al, 2003 ; Mustafa et al, 2004 ; Jaballah et al, 2010 ; Aktar et al, 2013 ; Kalloush et al, 2016 , 2019 ; Pitchai et al, 2018 ). Initial studies on MPMV identified a stretch of sequences at the 5′ end of the viral gRNA important for genome packaging that included sequences from R to the beginning of the gag open reading frame, including the major splice donor site (mSD; Figures 1A,B ; Harrison et al, 1995 ; Schmidt et al, 2003 ).…”