Norman H. Olson

Adding the Third Dimension to Virus Life Cycles: Three-Dimensional Reconstruction of Icosahedral Viruses from Cryo-Electron Micrographs

Viruses are cellular parasites. The linkage between viral and host functions makes the study of a viral life cycle an important key to cellular functions. A deeper understanding of many aspects of viral life cycles has emerged from coordinated molecular and structural studies carried out with a wide range of viral pathogens. Structural studies of viruses by means of cryo-electron microscopy and three-dimensional image reconstruction methods have grown explosively in the last decade. Here we review the use of cryo-electron microscopy for the determination of the structures of a number of icosahedral viruses. These studies span more than 20 virus families. Representative examples illustrate the use of moderate- to low-resolution (7- to 35-A) structural analyses to illuminate functional aspects of viral life cycles including host recognition, viral attachment, entry, genome release, viral transcription, translation, proassembly, maturation, release, and transmission, as well as mechanisms of host defense. The success of cryo-electron microscopy in combination with three-dimensional image reconstruction for icosahedral viruses provides a firm foundation for future explorations of more-complex viral pathogens, including the vast number that are nonspherical or nonsymmetrical.

Nucleocapsid and Glycoprotein Organization in an Enveloped Virus

Alphaviruses are a group of icosahedral, positive-strand RNA, enveloped viruses. The membrane bilayer, which surrounds the approximately 400 A diameter nucleocapsid, is penetrated by 80 spikes arranged in a T = 4 lattice. Each spike is a trimer of heterodimers consisting of glycoproteins E1 and E2. Cryoelectron microscopy and image reconstruction of Ross River virus showed that the T = 4 quaternary structure of the nucleocapsid consists of pentamer and hexamer clusters of the capsid protein, but not dimers, as have been observed in several crystallographic studies. The E1-E2 heterodimers form one-to-one associations with the nucleocapsid monomers across the lipid bilayer. Knowledge of the atomic structure of the capsid protein and our reconstruction allows us to identify capsid-protein residues that interact with the RNA, the glycoproteins, and adjacent capsid-proteins.

Assembly of a Tailed Bacterial Virus and Its Genome Release Studied in Three Dimensions

We present the first three-dimensional reconstruction of a prolate, tailed phage, and its empty prohead precursor by cryo-electron microscopy. The head-tail connector, the central component of the DNA packaging machine, is visualized for the first time in situ within the Bacillus subtilis dsDNA phage phi29. The connector, with 12- or 13-fold symmetry, appears to fit loosely into a pentameric vertex of the head, a symmetry mismatch that may be required to rotate the connector to package DNA. The prolate head of phi29 has 10 hexameric units in its cylindrical equatorial region, and 11 pentameric and 20 hexameric units comprise icosahedral end-caps with T=3 quasi-symmetry. Reconstruction of an emptied phage particle shows that the connector and neck/tail assembly undergo significant conformational changes upon ejection of DNA.

Structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor.

Two human rhinovirus serotypes complexed with two‐ and five‐domain soluble fragments of the cellular receptor, intercellular adhesion molecule‐1, have been investigated by X‐ray crystallographic analyses of the individual components and by cryo‐electron microscopy of the complexes. The three‐dimensional image reconstructions provide a molecular envelope within which the crystal structures of the viruses and the receptor fragments can be positioned with accuracy. The N‐terminal domain of the receptor binds to the rhinovirus ‘canyon’ surrounding the icosahedral 5‐fold axes. Fitting of molecular models into the image reconstruction density identified the residues on the virus that interact with those on the receptor surface, demonstrating complementarity of the electrostatic patterns for the tip of the N‐terminal receptor domain and the floor of the canyon. The complexes seen in the image reconstructions probably represent the first stage of a multistep binding process. A mechanism is proposed for the subsequent viral uncoating process.

Electron Microscopy Reveals the Nucleation Mechanism of Zeolite Y from Precursor Colloids

The growth of nanoscale crystals of zeolite Y from colloidal aluminosilicate gel particles has been investigated with high-resolution electron microscopy. Each amorphous gel particle nucleates only a single zeolite crystal, with nucleation beginning at the gel-solution interface (see scheme). Further growth of these nanocrystals is possible through the solution-mediated transport of framework building blocks.

Controlling and Switching the Morphology of Micellar Nanoparticles with Enzymes

Micelles were prepared from polymer-peptide block copolymer amphiphiles containing substrates for protein kinase A, protein phosphatase-1, and matrix metalloproteinases 2 and 9. We examine reversible switching of the morphology of these micelles through a phosphorylation-dephosphorylation cycle and study peptide-sequence directed changes in morphology in response to proteolysis. Furthermore, the exceptional uniformity of these polymer-peptide particles makes them amenable to cryo-TEM reconstruction techniques lending insight into their internal structure.

Putative receptor binding sites on alphaviruses as visualized by cryoelectron microscopy

The structures of Sindbis virus and Ross River virus complexed with Fab fragments from monoclonal antibodies have been determined from cryoelectron micrographs. Both antibodies chosen for this study bind to regions of the virions that have been implicated in cell-receptor recognition and recognize epitopes on the E2 glycoprotein. The two structures show that the Fab fragments bind to the outermost tip of the trimeric envelope spike protein. Hence, the same region of both the Sindbis virus and Ross River virus envelope spike is composed of E2 and is involved in recognition of the cellular receptor.

Structure of adeno-associated virus serotype 5.

ABSTRACT Adeno-associated virus serotype 5 (AAV5) requires sialic acid on host cells to bind and infect. Other parvoviruses, including Aleutian mink disease parvovirus (ADV), canine parvovirus (CPV), minute virus of mice, and bovine parvovirus, also bind sialic acid. Hence, structural homology may explain this functional homology. The amino acids required for CPV sialic acid binding map to a site at the icosahedral twofold axes of the capsid. In contrast to AAV5, AAV2 does not bind sialic acid, but rather binds heparan sulfate proteoglycans at its threefold axes of symmetry. To explore the structure-function relationships among parvoviruses with respect to cell receptor attachment, we determined the structure of AAV5 by cryo-electron microscopy (cryo-EM) and image reconstruction at a resolution of 16 Å. Surface features common to some parvoviruses, namely depressions encircling the fivefold axes and protrusions at or surrounding the threefold axes, are preserved in the AAV5 capsid. However, even though there were some similarities, a comparison of the AAV5 structure with those of ADV and CPV failed to reveal a feature which could account for the sialic acid binding phenotype common to all three viruses. In contrast, the overall surface topologies of AAV5 and AAV2 are similar. A pseudo-atomic model generated for AAV5 based on the crystal structure of AAV2 and constrained by the AAV5 cryo-EM envelope revealed differences only in surface loop regions. Surprisingly, the surface topologies of AAV5 and AAV2 are remarkably similar to that of ADV despite only exhibiting ∼20% identity in amino acid sequences. Thus, capsid surface features are shared among parvoviruses and may not be unique to their replication phenotypes, i.e., whether they require a helper or are autonomous. Furthermore, specific surface features alone do not explain the variability in carbohydrate requirements for host cell receptor interactions among parvoviruses.

DNA Poised for Release in Bacteriophage ø29

We present here the first asymmetric, three-dimensional reconstruction of a tailed dsDNA virus, the mature bacteriophage phi29, at subnanometer resolution. This structure reveals the rich detail of the asymmetric interactions and conformational dynamics of the phi29 protein and DNA components, and provides novel insight into the mechanics of virus assembly. For example, the dodecameric head-tail connector protein undergoes significant rearrangement upon assembly into the virion. Specific interactions occur between the tightly packed dsDNA and the proteins of the head and tail. Of particular interest and novelty, an approximately 60A diameter toroid of dsDNA was observed in the connector-lower collar cavity. The extreme deformation that occurs over a small stretch of DNA is likely a consequence of the high pressure of the packaged genome. This toroid structure may help retain the DNA inside the capsid prior to its injection into the bacterial host.

Regulation of the phage φ29 prohead shape and size by the portal vertex

Bacteriophage phi 29 of Bacillus subtilis packages its double-stranded DNA into a preformed prohead during morphogenesis. The prohead is composed of the scaffold protein gp7, the capsid protein pg8, the portal protein gp10, and the dispensable head fiber protein gp8.5. Our objective was to elucidate the phi 29 prohead assembly pathway and to define the factors that determine prohead shape and size. The structural genes of the phi 29 prohead were cloned and expressed in Escherichia coli individually or in combination to study form determination. The scaffold protein was purified from E. coli as a soluble monomer. In vivo and in vitro studies showed that the scaffolding protein interacted with both the portal vertex and capsid proteins. When the scaffold protein interacted only with the capsid protein in vivo, particles were formed with variable size and shape. However, in the presence of the portal vertex protein, particles with uniform size and shape were produced in vivo. SDS-PAGE analysis showed that the latter particles contained the proteins of the scaffold, capsid, head fiber, and portal vertex. These results suggest that the scaffolding protein serves as the linkage between the portal vertex and the capsid proteins, and that the portal vertex plays a crucial role in regulating the size and shape of the prohead.