Summary auto-generated
Researchers used the baculovirus expression system to produce murine polyomavirus capsid-like particles containing different combinations of structural proteins (VP1, VP2, and VP3). The study demonstrates that VP1 alone is sufficient to form capsid-like particles when expressed in Spodoptera frugiperda insect cells, while VP2 and VP3 cannot form particles independently but successfully incorporate into particles when co-expressed with VP1. All recombinant particles assembled to the proper size (45-50 nm diameter) and packaged approximately 5 kilobase pairs of cellular DNA with histones. The recombinant particles exhibited different VP1 protein species patterns compared to native virions, with additional minor VP1 species observed in the recombinant preparations, likely due to differences in posttranslational modifications between insect and mouse cells. A competition assay revealed that particles containing VP1 and VP2 were most effective at inhibiting native polyomavirus infection in mouse cells, suggesting VP2 plays a role in early viral entry events. This work provides a valuable tool for studying polyomavirus structural protein functions without complications from viral early genes or infectious DNA.
Key findings
- VP1 is essential and sufficient for capsid-like particle assembly; VP2 and VP3 alone cannot form particles but incorporate when co-expressed with VP1
- All recombinant particles package cellular DNA (~5 kbp) and histones while maintaining native virion morphology and size (45-50 nm)
- VP1 protein in recombinant particles shows different posttranslational modification patterns than native virions, with more VP1 species detected by two-dimensional gel electrophoresis
- Recombinant particles containing VP1 and VP2 demonstrate the highest ability to compete with and inhibit native polyomavirus infection, implicating VP2 in early infection events
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Abstract
The genes encoding the structural proteins (VP1, VP2 and VP3) of murine polyomavirus were cloned into the p2Bac dual multiple cloning site vector, individually or jointly, and the corresponding proteins were expressed in Spodoptera frugiperda (Sf9) insect cells by cotransfecting Sf9 cells with the constructed vector and the linear DNA of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Recombinant capsid-like particles could be purified 5 days post-infection from Sf9 cells infected with AcMNPV-VP1, with or without the involvement of minor protein (VP2 or VP3). Although VP2 and VP3 alone could not generate recombinant particles, they became incorporated into these particles when expressed with VP1 in Sf9 cells. Recombinant particles with different polyomavirus structural protein(s) were obtained by using different combined expression of these proteins in Sf9 cells. Cellular DNA of 5 kbp in size was packaged in all of the recombinant particles, which showed the same diameter as that of native virions. Agarose gel electrophoresis indicated that DNA packaged in these recombinant particles had a different pattern than that of native virions. Two-dimensional gel electrophoresis of the VP1 species of recombinant particles showed more VP1 species than those of the native virions from mouse cells, and an additional species of VP1 when VP2 was co-expressed with VP1. The recombinant particles were also compared for their ability to compete for polyomavirus infection. The competition assay indicated that the recombinant particles containing VP2 were the most efficient in inhibiting the native polyomavirus infection of 3T6 cells.