Abstract
SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of ∼6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility.
Highlights
SV40, a member of polyomaviradae family, has a small doublestranded circular DNA genome of 5.2 kb
We have found that the virus-like particles (VLPs) are significantly less stable than the virus
In contrast to wild type SV40, which required both DTT and EGTA for disassociation, VLPs consisting of VP1, VP2 and VP3 completely dissociated by incubation with either DTT or EGTA alone [37]
Summary
SV40, a member of polyomaviradae family, has a small doublestranded circular DNA genome of 5.2 kb. The DNA is complexed with histones, forming a nucleosomal structure similar to cellular chromatin, referred to as a minichromosome. VP3 translation initiates from an internal AUG within the VP2 coding sequence utilizing the same translational frame Both proteins share 234 identical amino acids at their carboxy part, and are frequently referred to as VP2/ 3. A single molecule of VP2 or VP3 is tightly attached to each pentamer at its inward facing cavity, through a region close to the C-terminus of VP2/3 [3,4]. These and other findings indicate that VP15VP2/3 is the building block for SV40 capsid assembly. VP1 has a jelly-roll b-barrel structure [1,5], with extending N-terminal arm, that carries the DNA-binding domain [6], and ,60 amino acids long C-terminal arm
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