Principles of Virology. Jane Flint
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METHODS
Nanoconstruction with virus particles
Nanochemistry is the synthesis and study of well-defined structures with dimensions of 1 to 100 nm. Molecular biologists study nanochemistry, nanostructures, and molecular machines including the ribosome and membrane-bound signaling complexes. Icosahedral viruses are proving to be precision building blocks for nanochemistry. The icosahedral cowpea mosaic virus particle is 30 nm in diameter, and its atomic structure is known. Grams of particles can be prepared easily from kilograms of infected leaves, insertional mutagenesis is straightforward, and precise amino acid changes can be introduced. As illustrated in panel A of the figure, cysteine residues inserted in the capsid protein provide functional groups for chemical attachment of 60 precisely placed molecules, in this case, gold particles.
High local concentrations of attached chemical agents, coupled with precise placement, and the propensity of virus-like particles for self-organization into two- and three-dimensional lattices of well-ordered arrays of particles enable rather remarkable nanoconstruction. For example, the surface of the filamentous bacteriophage M13 can be patterned to carry separate binding sites for gold and cobalt oxide and assembled into nanowires to form the anodes of small lithium ion batteries. Remarkably, this bacteriophage also displays intrinsic piezoelectric properties, that is, the ability to generate an electric charge in response to mechanical deformation, and vice versa. The basis of this property is not fully understood, but modification of the sequence of the major protein to increase its dipole moment (figure, panel B) augmented the piezoelectric strength of the bacteriophage. Assembly of the modified M13 into thin films was exploited to build a piezoelectric generator that produced up to 6 mÅ of current and 400 mV of potential, sufficient to operate a liquid crystal display (see Movie 4.1: http://bit.ly/Virology_piezo). Virus particles also have considerable potential for the delivery of drugs and other medically relevant molecules (Volume II, Chapter 9).
Gold particles attached to cowpea mosaic virus. (A) Cryo-EM was performed on derivatized cowpea mosaic virus with a cysteine residue inserted on the surface of each of the 60 subunits and to which nanogold particles with a diameter of 1.4 nm were chemically linked. (Left) Difference electron density map obtained by subtracting the density of unaltered cowpea mosaic virus at 29 Å from the density map of the derivatized virus. This procedure reveals both the genome (green) and the gold nanoparticles. (Right) A section of the difference map imposed on the atomic model of cowpea mosaic virus. The positions of the gold indicate that it is attached at the sites of the introduced cysteine residues. Courtesy of M.G. Finn and J. Johnson, The Scripps Research Institute. (B) Increasing the piezoelectric strength of phage M13. Schematic side view of a segment of M13 containing 10 copies (3 of which are shown) of the helical major coat protein modified to contain four glutamine residues at its N terminus. The dipole moments (yellow arrows) are directed from the N terminus (blue, positive) to the C terminus (red, negative).
Viruses are not just for infections anymore! They will provide a rich source of building blocks for applications spanning the worlds of molecular biology, materials science, and medicine.
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The internal components of influenza A virus particles differ radically: they comprise not a single nucleocapsid but multiple ribonucleoproteins, one for each of the 8 molecules of the segmented RNA genome present in an infectious virus particle (Appendix, Fig. 15). Furthermore, with the exception of terminal sequences, the RNA in these ribonucleoproteins is fully accessible to solvent, suggesting that the RNA is not sequestered in the interior of the ribonucleoprotein. The architectures of ribonucleoproteins released from influenza A virus particles determined by cryo-EM or scanning transmission EM tomography are consistent with such a model: the ribonucleoprotein comprises a double helix of NP molecules connected at one end by an NP loop, often with a molecule of the viral RNA polymerase bound at the other end (Fig. 4.8A). The RNA is bound along the exposed surfaces of the NP strands with some sequences in each RNA segment more tightly associated than others (Fig. 4.8B).
The examples presented above illustrate the diversity possible when viruses with simple helical symmetry possess an envelope. Exceptionally large examples include the (+) strand RNA virus potato virus Y, up to 900 nm in length, and bacterial inoviruses, some twice as long, that contain single-stranded DNA genomes. Nevertheless, helical viruses are limited in size. Because helical structures are “open,” some property other than symmetry must limit the size of helical viruses, perhaps the nature of their genomes (see Chapter 3) or susceptibility to shear forces.
Figure 4.6 Virus structures with helical symmetry. (A) Schematic illustration of a helical particle, indicating the individual subunits, their interaction to form a helical turn, the helix, and the helical parameters ρ