Opening and refolding of simian virus 40 and in vitro packaging of foreign DNA

Abstract

Simian virus 40 (SV40) can be disassembled under mild conditions by reducing disulfide bonds in the capsid and removing calcium ions. The nucleoprotein complexes formed, analyzed by electron microscopy, were circular and made up of 59 4 subunits, each with a diameter of about 10 nm. The complexes contained the viral DNA, histones, and the viral capsid proteins. The complexes had much-reduced infectivities compared with intact SV40. Addition of calcium ions to the disrupted virus caused the nucleoprotein complexes to refold into virus-like structures which sedimented at the same rate as intact SV40 and regained infectivity. Treatment of the disrupted SV40 with a high concentration of salt dissociated the viral proteins from the DNA. Lowering stepwise the salt concentration, removing the reducing agent, and adding calcium ions allowed structures to be reformed, and these structures sedimented, like SV40, at 240S and were infectious. The plaque-forming ability 2779

Evidence that SV40 VP1–DNA interactions contribute to the assembly of 40‐nm spherical viral particles

“…The main problem of the current VP1 assembly/ disassembly system is that morphologically normal 40-nm spherical particles are not formed under physiological conditions. The formation of 40-nm particles are observed only under high salt conditions as described above, and other diverse structures such as aggregates, 20-nm particles, and tubular structures are formed under physiological conditions instead (Colomar et al . 1993;Kanesashi et al .…”

“…VP1 self-assembles into virus-like particles (VLPs) when singly over-expressed in bacteria or in insect cells (Salunke et al . 1986;Colomar et al . 1993;Chang et al .…”

In Vitro Assembly of Virus-Like Particles and Their Applications

“…Bacteriophage Qβ VLPs were disassembled in 6 M urea [34], whereas 2.5 M urea were sufficient to denature hepatitis B core protein (HBc) VLPs [35]. Reducing and chelating agents can also be used in VLP disassembly [30,[36][37][38][39][40][41].…”