Structural puzzles in virology solved with an overarching icosahedral design principle

Viruses have evolved protein containers with a wide spectrum of icosahedral architectures to protect their genetic material. The geometric constraints defining these container designs, and their implications for viral evolution, are open problems in virology. The principle of quasi-equivalence is currently used to predict virus architecture, but improved imaging techniques have revealed increasing numbers of viral outliers. We show that this theory is a special case of an overarching design principle for icosahedral, as well as octahedral, architectures that can be formulated in terms of the Archimedean lattices and their duals. These surface structures encompass different blueprints for capsids with same numbers of structural proteins, as well as for capsid architectures formed from a combination of minor and major capsid proteins, and are conserved within viral lineages. They also apply to other icosahedral structures in nature, and offer alternative designs for man-made materials and nanocontainers in bionanotechnology

Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures.

Assembly of bacteriophage P22 procapsids requires the participation of approximately 300 molecules of scaffolding protein in addition to the 420 coat protein subunits. In the absence of the scaffolding, the P22 coat protein can assemble both wild-type-size and smaller size closed capsids. Both sizes of procapsid assembled in the absence of the scaffolding protein have been studied by electron cryomicroscopy. These structural studies show that the larger capsids have T = 7 icosahedral lattices and appear the same as wild-type procapsids. The smaller capsids possess T = 4 icosahedral symmetry. The two procapsids consist of very similar penton and hexon clusters, except for an increased curvature present in the T = 4 hexon. In particular, the pronounced skewing of the hexons is conserved in both sizes of capsid. The T = 7 procapsid has a local non-icosahedral twofold axis in the center of the hexon and thus contains four unique quasi-equivalent coat protein conformations that are the same as those in the T = 4 procapsid. Models of how the scaffolding protein may direct these four coat subunit types into a T = 7 rather than a T = 4 procapsid are presented

Solution x-ray scattering-based estimation of electron cryomicroscopy imaging parameters for reconstruction of virus particles.

Structure factor amplitudes and phases can be computed directly from electron cryomicroscopy images. Inherent aberrations of the electromagnetic lenses and other instrumental factors affect the structure factors, however, resulting in decreased accuracy in the determined three-dimensional reconstruction. In contrast, solution x-ray scattering provides absolute and accurate measurement of spherically averaged structure factor amplitudes of particles in solution but does not provide information on the phases. In the present study, we explore the merits of using solution x-ray scattering data to estimate the imaging parameters necessary to make corrections to the structure factor amplitudes derived from electron cryomicroscopic images of icosahedral virus particles. Using 400-kV spot-scan images of the bacteriophage P22 procapsid, we have calculated an amplitude contrast of 8.0 +/- 5.2%. The amplitude decay parameter has been estimated to be 523 +/- 188 A2 with image noise compensation and 44 +/- 66 A2 without it. These results can also be used to estimate the minimum number of virus particles needed for reconstruction at different resolutions

Mechanism of scaffolding-directed virus assembly suggested by comparison of scaffolding-containing and scaffolding-lacking P22 procapsids.

Assembly of certain classes of bacterial and animal viruses requires the transient presence of molecules known as scaffolding proteins, which are essential for the assembly of the precursor procapsid. To assemble a procapsid of the proper size, each viral coat subunit must adopt the correct quasiequivalent conformation from several possible choices, depending upon the T number of the capsid. In the absence of scaffolding protein, the viral coat proteins form aberrantly shaped and incorrectly sized capsids that cannot package DNA. Although scaffolding proteins do not form icosahedral cores within procapsids, an icosahedrally ordered coat/scaffolding interaction could explain how scaffolding can cause conformational differences between coat subunits. To identify the interaction sites of scaffolding protein with the bacteriophage P22 coat protein lattice, we have determined electron cryomicroscopy structures of scaffolding-containing and scaffolding-lacking procapsids. The resulting difference maps suggest specific interactions of scaffolding protein with only four of the seven quasiequivalent coat protein conformations in the T = 7 P22 procapsid lattice, supporting the idea that the conformational switching of a coat subunit is regulated by the type of interactions it undergoes with the scaffolding protein. Based on these results, we propose a model for P22 procapsid assembly that involves alternating steps in which first coat, then scaffolding subunits form self-interactions that promote the addition of the other protein. Together, the coat and scaffolding provide overlapping sets of binding interactions that drive the formation of the procapsid

Structural Localization of the E3 Glycoprotein in Attenuated Sindbis Virus Mutants

We have determined the three-dimensional structures of the wild-type Sindbis virus and two of its mutants that retain the E3 sequence within PE2. Using difference imaging between these mutants and the wild-type virus, we have assigned a location for the 64-amino-acid sequence corresponding to E3 in the mutant spike complex. In the wild-type virus, the spike is composed of an E1-E2 heterotrimer. The E3 protein was found to protrude midway between the center of the spike complex and the tips. Based on these results and the work of others, we propose a distribution for the functional domains of the spike proteins within the structure of wild-type Sindbis virus. Within the structure of the virus, the E1 domains form the central portion of the spike complex, while the tips are formed by the E2 domains that flare out from the center of the complex. The structural similarity between these Sindbis virus mutants and Ross River virus suggests that E3 may also be present in the latter, which is also a member of the Alphavirus genus