Principles of Virology. Jane Flint
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Portion of the cryo-EM difference map corresponding to the surface of one icosahedral face of the capsid. The crystal structures of the penton base (yellow) and the hexons (green, cyan, blue, and magenta at different positions) at appropriate resolution were docked within the cryo-EM density at 6-Å resolution. The cryo-EM density that does not correspond to these structural units (the difference map) is shown in red. At this resolution, the difference map revealed four trimeric structures located between neighboring hexons and three bundles of coiled-coiled α-helices. Both assemblies are now known to be formed by cement protein IX. Adapted from Saban SD et al. 2006. J Virol 80:12049–12059, with permission. Courtesy of Phoebe Stewart, Vanderbilt University Medical Center.
Helical Structures
The nucleocapsids of some enveloped animal viruses, as well as certain plant viruses and bacteriophages, are rod-like or filamentous structures with helical symmetry. Helical symmetry is described by the number of structural units per turn of the helix, the axial rise per unit, and the pitch of the helix (Fig. 4.6A). A characteristic feature of a helical structure is that any volume can be enclosed simply by varying the length of the helix. Such a structure is said to be open. In contrast, capsids with icosahedral symmetry (described below) are closed structures with fixed internal volume.
From a structural point of view, the best-understood helical nucleocapsid is that of tobacco mosaic virus, the very first virus to be identified. The virus particle comprises a single molecule of (+) strand RNA, about 6.4 kb in length, enclosed within a helical protein coat (Fig. 4.6B; see also Fig. 1.9). The coat is built from a single protein with an extended shape. Repetitive interactions among coat protein subunits form disks, which in turn assemble as a long, rod-like, right-handed helix. In the interior of the helix, each coat protein molecule binds three nucleotides of the RNA genome. The coat protein subunits therefore engage in identical interactions with one another and with the genome, allowing the construction of a large, stable structure from multiple copies of a single protein.
The particles of several families of animal viruses with (−) strand RNA genomes, including filoviruses, paramyxoviruses, rhabdoviruses, and orthomyxoviruses, contain internal structures with helical symmetry that are encased within an envelope. In all cases, these structures contain an RNA molecule, many copies of an RNA-packaging protein (designated NP or N), and the viral RNA polymerase and associated enzymes responsible for synthesis of mRNA and viral genomes. Despite common helical symmetry and similar composition, the internal components of these (−) strand RNA viruses exhibit considerable diversity in morphology and organization. For example, the nucleocapsids of the filovirus Zaire ebolavirus and the paramyxovirus Sendai virus are long, filamentous structures in which the NP proteins, like the tobacco mosaic virus coat protein, make regular interactions with the RNA genome. In contrast, the nucleocapsids of rhabdoviruses such as vesicular stomatitis virus are bullet shaped (Fig. 4.6C). Furthermore, an additional viral protein is essential to maintain their organization: vesicular stomatitis virus nucleocapsids released from within the envelope retain the dimensions and morphology observed in intact particles but become highly extended and filamentous once the matrix (M) protein is also removed (Fig. 12.21). X-ray crystallography of a ring-like N protein-RNA complex containing 10 molecules of the protein bound to RNA has revealed that each N protein molecule binds to 9 nucleotides of the RNA that is largely sequestered within cavities formed by the protein (Fig. 4.7). Furthermore, each N subunit makes extensive and regular contacts with neighboring N molecules, as predicted from first principles by Crick and Watson.
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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