Virus structure analysis with synchrotron radiation: methods and results.

John E Johnson

doi:10.1107/s0909049503023884

Home/Archive/PubMed

PubMed · 14646143

Virus structure analysis with synchrotron radiation: methods and results.

John E Johnson

View original source ↗Export metadata ↓

Abstract

Structural studies of viruses that are investigated as part of a program to understand molecular machines are described. Crystallography, solution X-ray scattering, electron microscopy and molecular virology were employed to investigate structure, assembly and maturation of RNA and dsDNA viruses. 240 copies of the RNA viral subunits (each with 650 amino acids) spontaneously assemble at pH 7 in a baculovirus expression system to form T = 4 icosahedral particles, 450 A in diameter. At pH 5 the particles condense to 410 A and the subunits auto-catalytically cleave at residue 570. 420 copies of the dsDNA viral subunits (281 amino acids each) assemble in an E. coli expression system to form T = 7 icosahedral particles, 450 A in diameter. At pH 4 the particles expand to 650 A diameter with the auto-catalytic formation of cross links between a lysine side chain and an asparagine side chain creating a concatenated set of 60 hexamer and 12 pentamer rings rendering the particle impervious to denaturation without protease treatment. Moderate- to high-resolution structures of the procapsids and capsids of these virus particles have been determined as well as cryoEM reconstructions of intermediate structures in order to define the driving force and the trajectories of these large-scale transitions.

Explore related subjects

Capsid Capsid Proteins Crystallography DNA, Viral Hydrogen-Ion Concentration Microscopy, Electron Nucleic Acid Conformation Nucleic Acid Denaturation RNA, Viral Synchrotrons X-Ray Diffraction Journal Article Research Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

John E Johnson. 2003-11-28. Virus structure analysis with synchrotron radiation: methods and results.. https://doi.org/10.1107/s0909049503023884

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2017

A directed evolution approach to select for novel Adeno-associated virus capsids on an HIV-1 producer T cell line.

A directed evolution approach was used to select for Adeno-associated virus (AAV) capsids that would exhibit more tropism toward an HIV-1 producer T cell line with the long-term goal of developing improved gene transfer vectors. A library of AAV variants was used to infect H9 T cells previously infected or uninfected by HIV-1 followed by AAV amplification with wild-type adenovirus. Six rounds of biological selection were performed, including negative selection and diversification after round three. The H9 T cells were successfully infected with all three wild-type viruses (AAV, adenovirus, and HIV-1). Four AAV cap mutants best representing the small number of variants emerging after six rounds of selection were chosen for further study. These mutant capsids were used to package an AAV vector and subsequently used to infect H9 cells that were previously infected or uninfected by HIV-1. A quantitative polymerase chain reaction assay was performed to measure cell-associated AAV genomes. Two of the four cap mutants showed a significant increase in the amount of cell-associated genomes as compared to wild-type AAV2. This study shows that directed evolution can be performed successfully to select for mutants with improved tropism for a T cell line in the presence of HIV-1.

Capsid↗

PubMed2014

Engineering adeno-associated viruses for clinical gene therapy.

Clinical gene therapy has been increasingly successful owing both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among these technologies, delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective and, in one recent case, have led to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers.

Capsid↗

PubMed2007

A precise packing sequence for self-assembled convex structures.

Molecular simulations of the self-assembly of cone-shaped particles with specific, attractive interactions are performed. Upon cooling from random initial conditions, we find that the cones self-assemble into clusters and that clusters comprised of particular numbers of cones (e.g., 4-17, 20, 27, 32, and 42) have a unique and precisely packed structure that is robust over a range of cone angles. These precise clusters form a sequence of structures at specific cluster sizes (a "precise packing sequence") that for small sizes is identical to that observed in evaporation-driven assembly of colloidal spheres. We further show that this sequence is reproduced and extended in simulations of two simple models of spheres self-assembling from random initial conditions subject to convexity constraints, including an initial spherical convexity constraint for moderate- and large-sized clusters. This sequence contains six of the most common virus capsid structures obtained in vivo, including large chiral clusters and a cluster that may correspond to several non-icosahedral, spherical virus capsids obtained in vivo. Our findings suggest that this precise packing sequence results from free energy minimization subject to convexity constraints and is applicable to a broad range of assembly processes.

Capsid↗