Assembly of fluorescent chimeric virus-like particles of canine parvovirus in insect cells

Abstract

Canine parvovirus (CPV) is a small non-enveloped ssDNA virus composed of the viral proteins VP1, VP2, and VP3 with a T=1 icosahedral symmetry. VP2 is nested in VP1 and the two proteins are produced by differential splicing of a primary transcript of the right ORF of the viral genome. The VP2 protein can be further proteolytically cleaved to form VP3. Previous studies have shown that VP1 and VP3 are unnecessary for capsid formation and consequently, that VP2 alone is sufficient for assembly. We have hypothesized that insertion of the enhanced green fluorescent protein (EGFP) at the N-terminus of VP2 could be carried out without altering assembly. To investigate the possibility to develop fluorescent virus-like particles (fVLPs) from such chimeric VP2 proteins, the corresponding fusion construct was abundantly expressed in insect cells. Confocal imaging indicated that the EGFP-VP2 fusion product was assembled to fluorescent capsid-like complexes. In addition, electron micrographs of purified EGFP-VP2 complexes showed that they displayed a very similar size and appearance when compared to VP2 VLPs. Further, immunolabelling of purified EGFP-VP2 VLPs showed the presence of EGFP within the structure. Fluorescence correlation spectroscopy (FCS) studies confirmed that fVLPs were very similar in size when compared to authentic CPV. Finally, feeding of mammalian cells susceptible to CPV infection with these fVLPs indicated that entry and intracellular trafficking could be observed. In summary, we have developed fluorescent virus-like nanoparticles carrying a heterologous entity that can be utilized as a visualization tool to elucidate events related to a canine parvovirus infection.