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

Area of Science:

• * Molecular Virology
• * Structural Biology
• * Protein Biochemistry

Background:

• * The Mason phenylo-melanoma virus (MPMV) precursor polypeptide Pr78^Gag is essential for assembling and packaging genomic RNA (gRNA) into new virus particles.
• * Understanding Pr78^Gag function is crucial for elucidating the mechanisms of viral RNA packaging.

Purpose of the Study:

• * To express and purify biologically active, recombinant full-length Pr78^Gag.
• * To investigate the in vitro and in vivo assembly of Pr78^Gag into virus-like particles (VLPs).
• * To assess the RNA encapsidation capability of VLPs formed by recombinant Pr78^Gag.

Main Methods:

• * Expression of recombinant Pr78^Gag (with and without His₆-tag) in bacterial and eukaryotic systems.
• * Purification of recombinant Pr78^Gag from soluble bacterial fractions.
• * In vitro and in vivo assembly assays to form VLPs.
• * RNA encapsidation assays using MPMV transfer vector RNA.

Main Results:

• * Recombinant Pr78^Gag was successfully expressed and purified.
• * The purified Pr78^Gag protein demonstrated an intrinsic ability to self-assemble into VLPs in vitro.
• * VLPs formed by recombinant Pr78^Gag in both prokaryotic and eukaryotic cells were observed.
• * VLPs produced in eukaryotic cells were capable of encapsidating MPMV transfer vector RNA, irrespective of the His₆-tag presence.

Conclusions:

• * Recombinant Pr78^Gag protein is biologically active and capable of forming VLPs.
• * The His₆-tag does not impede the expression, assembly, or RNA packaging function of Pr78^Gag.
• * This work provides a foundation for detailed studies on RNA-protein interactions in MPMV gRNA packaging.