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

Abstract

The simian virus 40 (SV40) particle is mainly composed of the major capsid protein termed VP1. VP1 self-assembles into virus-like particles (VLPs) of approximately 40 nm in diameter when over-expressed in bacteria or in insect cells, but purified VP1 does not form such a structure under physiological conditions, and thus, the mechanism of VP1 assembly is not well understood. Using a highly purified VP1 assembly/disassembly system in vitro , here we provide evidence that DNA is a factor that contributes to VP1 assembly into 40-nm spherical particles. At pH 5, for example, VP1 preferentially assembles into 40-nm particles in the presence of DNA, whereas VP1 assembles into tubular structures in the absence of DNA. Electron microscopic observations revealed that the concentration of DNA and its length are important for the formation of 40-nm particles. In addition, sucrose gradient sedimentation analysis and DNase I-sensitivity assays indicated that DNA of up to 2000 bp is packaged into the 40-nm particles under the conditions examined. We propose that DNA may facilitate the formation of 40-nm spherical particles by acting as a scaffold that increases the local concentration of VP1 and/or by acting as an allosteric effector that alters the structure of VP1.

Quantitative characterization of virus‐like particles by asymmetrical flow field flow fractionation, electrospray differential mobility analysis, and transmission electron microscopy

“…Packaging different nucleic acid lengths can affect the size of the VLP. For example, Tsukamoto et al (2007) showed that packaging lower molecular weight linear DNA in VLPs caused significant decreases in size. Similarly, studies of the packaging of RNA strands of varied length (Thomas et al, 2004) of the cowpea mosaic virus with ES-DMA found that encapsulation of longer RNA strands increased the average diameter of the virus by approximately 1 nm.…”

Quantum dot-induced viral capsid assembling in dissociation buffer

“…In the dissociation buffer, the disulfide bonds are broken by β-mercaptoethanol, and the Ca 2+ that stabilizes the VNP structures is chelated by ethylenediaminetetraacetic acid. 27,32 The low ionic strength and the lack of disulfide bonds lead to the collapse of the VNPs. This means that the concentrations of salt and Ca 2+ significantly contribute to the formation of VNPs.…”

“…[25][26][27][28] However, in this study, it was discovered that VP1 can be assembled into capsids when there are QDs in the dissociation buffer and that the QDs are encapsulated in the SV40 capsids. Analysis of the transmission electron microscopy (TEM), dynamic light scattering (DLS), sucrose density gradient centrifugation (SDGC), and cryo-electron microscopy (cryo-EM) single particle reconstruction data verified this result.…”

“…It is thought that the interaction between VP1 and DNA drives the virus assembly and DNA encapsulation. 26,27,34 The strong interaction between VP1 and MPA-QDs may play a role similar to that of the interaction between VP1 and DNA, and this interaction may act as the driving force behind the assembly of QD VNPs encapsulated in viral capsids. It was reported that the amino acids histidine, cysteine, methionine, and tryptophan have high affinity with cadmium selenide/zinc sulfide QDs, 36 so it is supposed that these amino acids of VP1 should play roles in triggering the assembly between VP1 and QDs.…”

“…Like DNA, the strong interaction of VP1 and QD cause the binding of VP1 to the QDs, which act as scaffolds that increase local concentrations of VP1 and as an allosteric effector that alter the tertiary structure of VP1. 27,34 The strong interaction between VP1 and QDs causes the binding of the VP1 proteins to the QDs, and this interaction results in the conformational change of VP1, which plays the similar role as the high concentrations of salt and Ca 2+ . 24,28 The tight binding of VP1 with QDs stabilizes the VNP structures.…”