High Cooperativity of the SV40 Major Capsid Protein VP1 in Virus Assembly

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

SV40 MORPHOGENESIS: PATHWAY AND EFFECT ON THE STRUCTURE OF SV40 CHROMATIN (VIRUS ASSEMBLY, PROTEIN-DNA INTERACTIONS, NUCLEOPROTEIN COMPLEX, TEMPERATURE-SENSITIVE MUTANT)

The Simian Virus 40 (SV40) morphogenetic pathway was studied by analyzing the nucleoprotein complexes assembled in cells infected with a wild-type SV40 virus (wt776) and mutants temperature-sensitive in virus assembly: tsB and tsC. The tsB and tsC mutations map to the gene coding for the major capsid protein VP1. At the nonpermissive temperature (40(DEGREES)C), the 75 S viral chromatin accumulates in tsC-infected cells whereas, in tsB-infected cells, the viral chromatin associates with the capsid proteins VP1, VP2, and VP3 to form semiassembled virus particles (SAP) which sediment heterogeneously from 100 to 160 S. The structural and biochemical properties of the tsB mutants support the hypothesis that SV40 assembly proceeds by the gradual addition of the capsid proteins onto chromatin. Further characterization of the tsB mutants by pulse-chase kinetic studies and temperature shift experiments revealed a critical relationship between VP1 structure and its function in the capsid shell polymerization process. On the other hand, the tsC mutants seem to be defective in the initiation of shell assembly on viral chromatin. Micrococcal and DNAase I digestion analyses were performed on tsC chromatin, wt776 chromatin and tsB SAP to study the organization of histones on SV40 DNA and the possible effect of virus assembly on this organization. Micrococcal nuclease digestion analysis revealed that tsC chromatin assembled at 40(DEGREES)C possesses a shorter average nucleosomal repeat length (179 base pairs) compared to that observed for wt776 chromatin (195 base pairs) or tsB SAP (198 base pairs). Further studies demonstrated that the shorter tsC chromatin repeat length observed results from an alteration in a VP1 function specific to tsC mutants. These findings provide the first genetic evidence involving a nonhistone protein in the determination of the nucleosomal repeat length in nucleohistones, and lead to a model in which the capsid proteins play a role in a nucleosomal rearrangement process that functions not only in virus assembly but also in controlling viral gene expression. Further characterization of the SV40 nucleoprotein complexes by DNAase I digestion analysis indicated preferred nucleosome positioning sites on SV40 DNA. Some of these sites were shared by tsC, wt and tsB nucleoprotein complexes

Optimization of a BiCMOS integrated tranducer for self-compensated capacitive pressure sensor

International audienceTwo new pressure sensor demonstrators have been designed and mounted using silicon/Pyrex capacitive sensing cells and analog-digital BiCMOS transducers. A ratiometric scheme has been used to self-compensate thermal drifts and nonlinearities. The first demonstrator implemented with four chips is characterized by a relative sensitivity close to-1.85%/bar, a nonlinearity in the order of 1.1%FS and an offset thermal coefficient smaller than 20 ppm/°C. The second one has been implemented with two integrated chips. It consists, on the one hand, of two pairs of converters and counters integrated in the same chip, and on the other hand, of a reference and measurement capacitors into the same sensing cell. This configuration permits to reduce stray capacitors by a factor two but also provokes interference between oscillators. Characterization and PSPICE modeling have permitted to understand, to localize causes and then to propose some solutions to avoid and/or minimize these problems