Assembly of double-layered virus-like particles in mammalian cells by coexpression of human rotavirus VP2 and VP6

Abstract

Development in mammalian cells of a recombinant expression system that mimics the rotavirus capsid assembly process would be advantageous for studying the structural requirements for particle formation. To this end, we investigated the ability of a recombinant vaccinia virus system to produce double-layered viruslike particles. The genes coding for VP2 and VP6 proteins of the human rotavirus strain Wa were cloned and used to generate recombinant vaccinia viruses. Metabolic labelling of CV-1 cells infected with these recombinant viruses followed by immunoprecipitation with a polyclonal antiserum directed to Wa virus showed that VP2 and VP6 were efficiently expressed. The recombinant proteins were similar in size and immunoreactivity to authentic rotavirus proteins. Biochemical and electron microscopy analyses demonstrated that simultaneous expression of VP2 and VP6 in mammalian cells resulted in the formation of intracellular spherical particles resembling double-layered rotavirus particles.

Comparative immunogenicity in mice of rotavirus VP6 tubular structures and virus-like particles

“…Figure 1A shows sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel with the identified RV VP6 (45 kDa) and VP2 (102 kDa) bands of purified products. In concordance with the work of others 30 we also detected an additional cleavage product of VP2 with an apparent molecular weight of ~90 kDa. Oligomeric structures including rVP6 tubules (~0.2-1.5 μm in length) with hexagonal subunit pattern and dl2/6-VLPs (~65 nm in diameter) were confirmed under electron microscopy (EM) (Fig.…”

Rotavirus VP6 preparations as a non-replicating vaccine candidates

“…It has been shown by several studies that the structural proteins (VP2, VP6, VP7, and VP4) of RV can be expressed in different prokaryotic and eukaryotic expression systems and form SLPs (VP2), DLPs (VP2 and VP6), and TLPs (VP2, VP6, and VP7 or VP2, VP6, VP7, and VP4) [132][133][134][135][136][137][138][139][140][141][142][143][144][145][146][147][148]. Furthermore, expressing VP6 alone in vitro [14,29] can form spherical, tubular, and trimer structures similar to VP6 purified from the RVs [31].…”

Characterization and protective efficacy in an animal model of a novel truncated rotavirus VP8 subunit parenteral vaccine candidate

“…In the case of rotavirus, VP2/6/7 or VP2/6/4/7 triple-layered virus-like particles (VLPs) [19,20], VP2/6 doublelayered VLPs [21] and VP6 subunit protein [22,23] eukaryotic cells have proven to be effective against rotavirus induced diarrhea or virus shedding in small animal models. However, the eukaryotic expression of VLPs need simultaneous expression of several recombinant rotavirus proteins in a single cell, which poses challenges for vaccine bioprocessing and bioanalytics [24].…”