Analysis of Simian Virus 40 Chromatin Structure by Cryo-Electron Tomography

Abstract

Simian virus 40 (SV40) is a small (~45nm), non-enveloped, DNA virus with an icosahedral capsid. Viral DNA associates with histones in the nucleus of infected cells to form a chromatinized, supercoiled, circular minichromosome. The SV40 capsid proteins assemble around this minichromosome to form new virions. The capsid contains 72 pentamers of VP1 protein arrayed on a T=7d icosahedral lattice. Single molecules of VP2/3 protein are associated with the inner surface of all or most VP1 pentamers, potentially linking to the viral chromatin. The icosahedral symmetry of the capsid has allowed its structure to be determined at high resolution by particle averaging and crystallographic methods [1]. However, the organization of the viral chromatin -which is not expected to conform to this symmetry -has not been determined.To study the chromatin structure of individual SV40 particles, we collected single-axis tilt series of plunge-frozen virus particles across an angular range of -70 to +70 degrees on a 120kV Tecnai T12 TEM equipped with an energy filter operating in zero-loss mode. Images were recorded at 2° increments at a dose of 1 e -/Å 2 per image. Three-dimensional reconstructions of these tilt series were computed using IMOD [2]. Reconstructions were denoised by nonlinear anisotropic diffusion filtering. The average in-plane resolution of the reconstructions was calculated by the NLOO method to be 5 nm [3], sufficient to observe nucleosome structures.Individual virions were extracted from tomograms for further analysis. Punctate densities, consistent in size and appearance with nucleosomes, are visible inside the capsids (Fig. 1A). The most prominent viral component was the capsid shell, whose T=7d icosahedral symmetry was clearly visible in individual subtomograms (Fig. 1B) and was further enhanced by particle averaging (Fig. 1C). As expected, averaging revealed no symmetrically distributed internal densities; rather, it produced a low uniform level of internal density. These findings are consistent with earlier data suggesting that viral chromatin is not icosahedrally ordered [4]. Quantifying the nucleosome densities present in individual particles revealed them to be variable in number, with an average of 19 +/-2.5 per particle (Fig. 2). The tomograms also suggest the presence of contacts between some internal chromatin densities and the capsid shell. Ongoing efforts are focused on better defining these putative capsid-chromatin links, as well as the organization of encapsidated minichromasomes.

Genome packaging within icosahedral capsids and large-scale segmentation in viral genomic sequences

“…For SV40 commonly the lower numbers for nucleosomes, ~20-26. The packaging with the such numbers of nucleosomes cannot be in a complete correspondence with the icosahedral symmetry and a part of randomization in nucleosome positioning within capsid observed experimentally (Keller et al, 2009;Saper et al, 2013;Hurdiss et al, 2016) can be attributed to such incomplete correspondence. By analogy with the cryo-EM asymmetric reconstruction for MS2, the results by Saper et al (2013) and Hurdiss et al (2016) indicate the presence of internal fraction in DNA packaging.…”

“…Estimating the volume of nucleosome without H1 as about 440 nm 3 and the filling factor as 0.6, the number of nucleosomes within capsid may amount to 20 that is close to the experimentally observed number. Keller et al (2009) studied SV40 chromatin structure via cryo-electron tomography, however, the organization of the viral chromatin has not been determined. Saper et al (2013) using small-angle X-ray scattering proved that minichromosomal density increases toward the center of the capsid.…”