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

Canine parvovirus (CPV) is an autonomous, non-enveloped single stranded DNA virus with a diameter of 26 nm. The icosahedral T = 1 virion contains 60 protein subunits composed of three different polypeptide chains designated VP1, VP2, and VP3 [1-7]. VP1 is identical to VP2, but has 154 additional N-terminal amino acid residues. The VP3 protein is proteolytically cleaved from VP2 by removal of about 12 to 15 amino acids from the N-terminus [1,8]. The VP2 protein constitutes most of the capsid surface while VP1 represents only a small portion of the capsid composition. It has been shown that VP2 can assemble into capsid-like structures [9] and that the structure of empty CPV capsids had the first 37 residues not resolved structurally [9]. These structural proteins share a conserved β-barrel core domain that contains an eight-stranded, anti-parallel β-barrel motif consisting of two β-sheets in standard BIDG and CHEF arrangements common to many viral capsid proteins [10]. This domain accounts for one third of the amino acid content of each polypeptide. The other two thirds of the polypeptide sequence consist of four large loop insertions that form the surface of the virion.Viral structures have been mainly characterized by X-ray crystallography and electron microscopy. Single molecule detection techniques have arisen for characterization of macromolecules moving persistently in non-denaturing physiological conditions. One such emerging method is fluorescence correlation spectroscopy (FCS) [11-14]. FCS characterizes interactions and molecular structures through the dynamic processes of molecules in solution. Statistical information is extracted from the averaged fluorescence intensity fluctuations of fluorescent molecules diffusing through a small measuring volume of less than one femtoliter [15,16].In the present study, 14, 23 and 40 N-terminal amino acid deletions of the VP2 protein were fused to the C-terminus of EGFP. The corresponding proteins were produced in baculo