Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly

Abstract

A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2" ions and the removal of chelating and reducing agents. Budgerigar fledgling disease virus (BFDV), the first avian member of the polyomavirus family, was found to cause an acute disease in fledgling budgerigars (3, 5, 23, 24). BFDV is classified as a polyomavirus on the basis of its morphology, its resistance to organic solvents, and the facts that its virions have a buoyant density of 1.34 g/ml and its DNA is supercoiled and double stranded with a molecular weight of 330,000 (5, 16, 28). BFDV also has the capability to transform nonpermissive primary hamster embryo fibroblasts, an ability common to polyomaviruses (28). Many similarities were found when the genome of BFDV, composed of 4,980 bp (42), was compared with the genomes of both murine polyomavirus and simian virus 40 (SV40) (38). The genomes of these three viruses can be divided into early and late coding regions. The early region of BFDV codes for large and small T antigens similar to those of SV40 and murine polyomavirus. Murine polyomavirus also codes for a middle T antigen, while BFDV and SV40 do not (38,47). The late region in all three viruses codes for the three structural proteins VP1, VP2, and VP3. In BFDV the molecular masses of these structural proteins are 42, 39, and 29 kDa, respectively (22). Previous studies have shown that the major capsid protein VP1, in both murine polyomavirus and SV40, can be differentially modified during translation in several ways (31, 32), including phosphorylation (1, 2, 4, 18, 19, 29, 39). Recently it was reported that the VP1 protein of BFDV can also be posttranslationally modified and that the VP1 protein consists of five distinct subspecies, with two being phosphorylated (22). Schmidt et al. (45) demonstrated that the minor capsid protein VP2 was modified by myristoylation. A recombinant system has been established for the major capsid protein VP1 of murine polyomavirus (27). This recombinant system has generated large quantities of the VP1 protein, which have been used in numerous studies (10, 11, 21, 36, 37, 43, 44). The current study used a similar strategy for the

Disulfide bonds stabilize JC virus capsid‐like structure by protecting calcium ions from chelation

“…2C) with hemagglutination activity. These results are similar to previous reports [16,18,19,21] and indicate that disulfide bonds and calcium ions are involved in stabilizing the VLP, and that calcium ions but not disulfide bonds are required for capsid assembly.…”

“…The supernatant of the yeast cell lysate containing VP1 protein was subjected to 20% sucrose cushion centrifugation at 35 000 rpm for 3 h in a SW41 Ti rotor (Beckman). The pellet was resuspended in Tris buffer and diluted to a density of 1.20 g/cm 3 , then layered onto a five‐step CsCl velocity gradient consisting of 2 ml of 1.20, 1.25, 1.29, 1.32 and 1.35 g/cm 3 CsCl solutions [20,21]. After centrifugation at 35 000 rpm for 24 h, the solution was fractionated.…”

“…Samples containing purified VLPs and capsomeres were mounted on formvar‐coated grids and stained with 1% uranyl acetate [19–21]. Specimens were examined with a Joel 2000‐CX electron microscope operated at 80 kV.…”

“…Either a reducing agent or a metal ion chelator alone can not disrupt the capsid particle [17,19]. Instead, capsomeres can be reassembled into a capsid‐like structure in the presence of calcium ions alone without the involvement of disulfide bonds [18,20,21]. Therefore, the role of disulfide bonds in polyomavirus assembly needs to be further clarified.…”

Identification of a DNA encapsidation sequence for human polyomavirus pseudovirion formation

“…The primers were constructed with Bam HI sites immediately 5 H to the start or 3 H to the stop codon to facilitate subsequent manipulation. The ampli®ed DNA fragments were digested with Bam HI, puri®ed using agarose gel electrophoresis and Magic PCR Preps columns (Promega, Madison, WI), and cloned into Bgl II-digested prokaryotic expression vector DpFlag [Chang et al, 1993;Rodgers et al, 1994] to express the non-fusion VP1 mutant proteins. The constructs were transformed into E. coli JM109 cells and the proteins were induced at an early logarithmic phase by the addition of 0.5 mM (®nal concentration) isopropyl-b-D-thiogalactopyranoside (IPTG) for 4 hr at 308C.…”