Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

Abstract

Fluorescence correlation spectroscopy (FCS) monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nanoparticles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids) were fused to the C-terminus of the enhanced green fluorescent protein (EGFP). The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs). The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.

Display of multiple proteins on engineered canine parvovirus-like particles expressed in cultured silkworm cells and silkworm larvae

“…Before further modifications of the location of SpT, we confirmed the VLP formation of the self-assembled SpT-VP2 in TEM ( Figure 1F ) and investigated the possible protein display by immuno-TEM using anti-His6 for SpC-EGFP ( Figure 1G ). The intact VLP structure and distribution (20–30 nm) were validated ( Figure 1H ) in this study, consistent with previous studies ( Choi et al, 2000 ; Gilbert et al, 2006 ). This result provides direct evidence that VP2-derived SpT-SpC partner can be successfully expressed in silkworm-BEVS and be realized as a VLP display system.…”

“…The VLP formation of CPV can be obtained via the self-assembly of capsid VP2 protein in various protein expression systems, including BEVS with cultured insect Sf9 cells, silkworm Bm5 cells, silkworm larvae, and pupae ( Choi et al, 2000 ; Feng et al, 2014 ; Jin et al, 2016 ; Chang et al, 2020 ). Previous literature has also found that the N-terminal genetically fused GFP can be used as a protein display strategy ( Gilbert et al, 2006 ). Thus, as a continuous effort for our previous SpyBEVS ( Xu et al, 2019 ), we designed several SpT-SpC chemistry constructs at terminal regions to establish protein display on CPV-LPs in silkworm-BEVS (SpyVLP-BEVS), which are illustrated in Figures 1A, B .…”

“…The low display level is consistent with a previous study of N-terminal EGFP-fused VP2-derived VLP since only 20% (12 out of 60, N-terminal fused EGFP protrudes through the 5-fold axis cannon structures) display rate was simulated. A 16.7% (10/60) could be experimentally achieved on the VLP surface ( Gilbert et al, 2006 ). Before further modifications of the location of SpT, we confirmed the VLP formation of the self-assembled SpT-VP2 in TEM ( Figure 1F ) and investigated the possible protein display by immuno-TEM using anti-His6 for SpC-EGFP ( Figure 1G ).…”

Study of canine parvovirus evolution: comparative analysis of full-length VP2 gene sequences from Argentina and international field strains

“…Amino acid position 101, although also presenting a fixed substitution (I101T), was identified as being under negative selection pressure, probably due to the observed existence of two different triplets codifying for the same aa (data not shown). An interesting observation related to the tridimensional structure of VP2 is that positions 87, 101, and 555, although outside the GH loop, are in an exposed region of the protein [48,49].…”

Parvovirus Induced Alterations in Nuclear Architecture and Dynamics

“…The diffusion dynamics were examined using photoactivable GFP (PAGFP) fused N-terminally with the major capsid protein VP2. It is known that such a fusion allows for assembly of VLPs [49] . Also, in the case of another parvovirus, the minute virus of mice, the capsid proteins enter the nuclei only in trimeric form [50] .…”