Extracellular self-assembly of virus-like particles from secreted recombinant polyoma virus major coat protein

Abstract

Mouse polyoma virus major coat protein (VP1) expressed from a recombinant baculovirus is efficiently transported to infected cell nuclei and assembles into protein nanospheres morphologically similar to natural capsids. The nanospheres readily combine with plasmid DNA to form a hybrid gene therapy agent known as virus-like particles (VLPs). To facilitate large-scale production of VLPs free from cellular contaminants, the use of stable Drosophila cell lines expressing either wild-type protein, or VP1 tagged with a secretion signal for targeting to the extracellular medium, was investigated. Both wild-type and tagged VP1 expressed at 2-4 mg VP1/litre of culture. As expected, the wild-type protein self-assembled into VLPs. The tagged VP1 was efficiently secreted to the extracellular medium but was also glycosylated, unlike wild-type VP1. Despite this fact, a small fraction of the recombinant secreted protein assembled into VLP-like structures that had altered disulphide bonding, but were still biologically active. These results demonstrate the considerable tolerance in the nanosphere assembly to structural (i.e. aberrant glycosylation) and environmental (i.e. extracellular medium vs. nuclear milieu) changes. Thus, with modifications to improve nanosphere assembly, the secretion method could be adapted to large-scale preparation of VLPs, providing significant advantages over current methods of production of the vector.

Leflunomide Inhibition of BK Virus Replication in Renal Tubular Epithelial Cells

“…However, our data implicate inhibition of virion assembly and release, with altered nuclear distribution of VP1 and LT-ag. This could be caused by modified signal transduction pathways or posttranslational modifications of VP1, i.e., phosphorylations, acetylation, and methylation (32). Furthermore LEF-A may inhibit Ca 2ϩ mobilization (46), which plays an important role in viral assembly (33).…”

Expression and Purification of Porcine Rotavirus Structural Proteins in Silkworm Larvae as a Vaccine Candidate

“…Therefore, these results suggest that introducing N -glycosylation of VP6 via the silkworm glycosylation pathway impedes the formation of VP6-derived VLPs. Previous reports indicate that when the VP1 of mouse polyomavirus is expressed with aberrant N -glycosylation in insect cells, VP1 remains in a monomeric form and only little formation of VLPs takes place [ 34 ]. It has also been reported earlier that although, adeno-associated virus type 2 has three putative N -glycosylation sites, its capsid protein remains unglycosylated [ 35 ].…”