Mark J. Young

Host–guest encapsulation of materials by assembled virus protein cages

Self-assembled cage structures of nanometre dimensions can be used as constrained environments for the preparation of nanostructured materials, and the encapsulation of guest molecules, with potential applications in drug delivery and catalysis. In synthetic systems the number of subunits contributing to cage structures is typically rather small,. But the protein coats of viruses (virions) commonly comprise hundreds of subunits that self-assemble into a cage for transporting viral nucleic acids. Many virions, moreover, can undergo reversible structural changes that open or close gated pores to allow switchable access to their interior. Here we show that such a virion — that of the cowpea chlorotic mottle virus — can be used as a host for the synthesis of materials. We report the mineralization of two polyoxometalate species (paratungstate and decavanadate) and the encapsulation of an anionic polymer inside this virion, controlled by pH-dependent gating of the virions pores. The diversity in size and shape of such virus particles make this a versatile strategy for materials synthesis and molecular entrapment.

Inorganic–Organic Nanotube Composites from Template Mineralization of Tobacco Mosaic Virus

The use of biological molecules, assemblies and systems in the development of inorganic materials synthesis continues to offer new and exciting alternatives to conventional synthetic strategies. Biological templates, such as protein cages, viroid capsules, bacterial rhapidosomes, S-layers, multicellular superstructures, biolipid cylinders, and DNA, have been utilized to direct the deposition, assembly, and patterning of inorganic nanoparticles and microstructures. In this paper, we report a new approach to the template-directed synthesis of inorganic±organic nanotubes using tobacco mosaic virus (TMV). TMV is a remarkably stable virion, remaining intact at temperatures up to 60 C and at pH values between 2 and 10. Each viral particle consists of 2130 identical protein subunits arranged in a helical motif around a single strand of RNA to produce a hollow protein tube, 300 18 nm in size, with a 4 nm-wide central channel. The internal and external surfaces of the protein consist of repeated patterns of charged amino acid residues, such as glutamate, aspartate, arginine, and lysine. In principle, these functionalities should offer a wide variety of nucleation sites for surface-controlled inorganic deposition, which, in association with the high thermal and pH stability, could be exploited in the synthesis of unusual materials such as high-aspect-ratio composites and protein-confined inorganic nanowires. Here we show that TMV is a suitable template for reactions such as co-crystallization (CdS and PbS), oxidative hydrolysis (iron oxides), and sol-gel condensation (SiO2) (Fig. 1).

Viruses from extreme thermal environments

Viruses of extreme thermophiles are of great interest because they serve as model systems for understanding the biochemistry and molecular biology required for life at high temperatures. In this work, we report the discovery, isolation, and preliminary characterization of viruses and virus-like particles from extreme thermal acidic environments (70–92°C, pH 1.0–4.5) found in Yellowstone National Park. Six unique particle morphologies were found in Sulfolobus enrichment cultures. Three of the particle morphologies are similar to viruses previously isolated from Sulfolobus species from Iceland and/or Japan. Sequence analysis of their viral genomes suggests that they are related to the Icelandic and Japanese isolates. In addition, three virus particle morphologies that had not been previously observed from thermal environments were found. These viruses appear to be completely novel in nature.

Paramagnetic viral nanoparticles as potential high-relaxivity magnetic resonance contrast agents

In order to compensate for the inherent high threshold of detectability of MR contrast agents, there has been an active interest in the development of paramagnetic nanoparticles incorporating high payloads of Gd3+ with high molecular relaxivities. Toward this end, the protein cage of Cowpea chlorotic mottle virus (CCMV), having 180 metal binding sites, is being explored. In vivo CCMV binds Ca2+ at specific metal binding sites; however, Gd3+ can also bind at these sites. Using fluorescence resonance energy transfer we have characterized the binding affinity of Gd3+ to the metal binding sites by competition experiments with Tb3+. The measured dissociation constant (Kd) for Gd3+ bound to the virus is 31 μM. The T1 and T2 relaxivities of solvent water protons in the presence of Gd3+‐bound CCMV were 202 and 376 mM−1 s−1, respectively, at 61 MHz Larmor frequency. The unusually high relaxivity values of the Gd3+–CCMV are largely a result of the nanoparticle virus size and the large number of Gd3+ ions bound to the virus. These preliminary results should encourage further investigations into the use of viral protein cages as a new platform for MR contrast agents. Magn Reson Med, 2005. Published 2005 Wiley‐Liss, Inc.

Assembly of viral metagenomes from yellowstone hot springs.

ABSTRACT Thermophilic viruses were reported decades ago; however, knowledge of their diversity, biology, and ecological impact is limited. Previous research on thermophilic viruses focused on cultivated strains. This study examined metagenomic profiles of viruses directly isolated from two mildly alkaline hot springs, Bear Paw (74°C) and Octopus (93°C). Using a new method for constructing libraries from picograms of DNA, nearly 30 Mb of viral DNA sequence was determined. In contrast to previous studies, sequences were assembled at 50% and 95% identity, creating composite contigs up to 35 kb and facilitating analysis of the inherent heterogeneity in the populations. Lowering the assembly identity reduced the estimated number of viral types from 1,440 and 1,310 to 548 and 283, respectively. Surprisingly, the diversity of viral species in these springs approaches that in moderate-temperature environments. While most known thermophilic viruses have a chronic, nonlytic infection lifestyle, analysis of coding sequences suggests lytic viruses are more common in geothermal environments than previously thought. The 50% assembly included one contig with high similarity and perfect synteny to nine genes from Pyrobaculum spherical virus (PSV). In fact, nearly all the genes of the 28-kb genome of PSV have apparent homologs in the metagenomes. Similarities to thermoacidophilic viruses isolated on other continents were limited to specific open reading frames but were equally strong. Nearly 25% of the reads showed significant similarity between the hot springs, suggesting a common subterranean source. To our knowledge, this is the first application of metagenomics to viruses of geothermal origin.

Virus Particles as Templates for Materials Synthesis

The interface between biology, chemistry, and materialsscience has provided inspiration for novel approaches to theformationofmaterials.Increasingly,organizedbiomoleculararchitectures are being used as templates for the precisepatterning of inorganic materials in a biomimetic approachto materials synthesis. For example bacterial S-layers,

Metagenomes from high-temperature chemotrophic systems reveal geochemical controls on microbial community structure and function.

The Yellowstone caldera contains the most numerous and diverse geothermal systems on Earth, yielding an extensive array of unique high-temperature environments that host a variety of deeply-rooted and understudied Archaea, Bacteria and Eukarya. The combination of extreme temperature and chemical conditions encountered in geothermal environments often results in considerably less microbial diversity than other terrestrial habitats and offers a tremendous opportunity for studying the structure and function of indigenous microbial communities and for establishing linkages between putative metabolisms and element cycling. Metagenome sequence (14–15,000 Sanger reads per site) was obtained for five high-temperature (>65°C) chemotrophic microbial communities sampled from geothermal springs (or pools) in Yellowstone National Park (YNP) that exhibit a wide range in geochemistry including pH, dissolved sulfide, dissolved oxygen and ferrous iron. Metagenome data revealed significant differences in the predominant phyla associated with each of these geochemical environments. Novel members of the Sulfolobales are dominant in low pH environments, while other Crenarchaeota including distantly-related Thermoproteales and Desulfurococcales populations dominate in suboxic sulfidic sediments. Several novel archaeal groups are well represented in an acidic (pH 3) Fe-oxyhydroxide mat, where a higher O2 influx is accompanied with an increase in archaeal diversity. The presence or absence of genes and pathways important in S oxidation-reduction, H2-oxidation, and aerobic respiration (terminal oxidation) provide insight regarding the metabolic strategies of indigenous organisms present in geothermal systems. Multiple-pathway and protein-specific functional analysis of metagenome sequence data corroborated results from phylogenetic analyses and clearly demonstrate major differences in metabolic potential across sites. The distribution of functional genes involved in electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, Fe, O2) control microbial community structure and function in YNP geothermal springs.

A human ferritin iron oxide nano-composite magnetic resonance contrast agent

Macrophages play important roles in the immunological defense system, but at the same time they are involved in inflammatory diseases such as atherosclerosis. Therefore, imaging macrophages is critical to assessing the status of these diseases. Toward this goal, a recombinant human H chain ferritin (rHFn)‐iron oxide nano composite has been investigated as an MRI contrast agent for labeling macrophages. Iron oxide nanoparticles in the form of magnetite (or maghemite) with narrow size distribution were synthesized in the interior cavity of rHFn. The composite material exhibited the R2 relaxivity comparable to known iron oxide MRI contrast agents. Furthermore, the mineralized protein cages are readily taken up by macrophages in vitro and provide significant T2* signal loss of the labeled cells. These results encourage further investigation into the development of the rHFn‐iron oxide contrast agent to assess inflammatory disease status such as macrophage‐rich atherosclerotic plaques in vivo. Magn Reson Med 60:1073–1081, 2008.

Viral capsids as MRI contrast agents.

Viral capsids have the potential for combined cell/tissue targeting, drug delivery, and imaging. Described here is the development of a viral capsid as an efficient and potentially relevant MRI contrast agent. Two approaches are outlined to fuse high affinity Gd3+ chelating moieties to the surface of the cowpea chlorotic mottle virus (CCMV) capsid. In the first approach, a metal binding peptide has been genetically engineered into the subunit of CCMV. In a second approach gadolinium‐tetraazacyclododecane tetraacetic acid (GdDOTA) was attached to CCMV by reactions with endogenous lysine residues on the surface of the viral capsid. T1 and T2 ionic relaxivity rates for the genetic fusion particle were R1 = 210 and R2 = 402 mM−1s−1 (R2 at 56 MHz) and for CCMV functionalized with GdDOTA were R1 = 46 and R2 = 142 mM−1s−1 at 61 MHz. The relaxivities per intact capsid for the genetic fusion were R1 = 36,120 and R2 = 69,144 mM−1s−1 (R2 at 56 MHz) and for the GdDOTA CCMV construct were R1 = 2,806 and R2 = 8,662 mM−1s−1 at 61 MHz. The combination of high relaxivity, stable Gd3+ binding, and large Gd3+ payloads indicates the potential of viral capsids as high‐performance contrast agents. Magn Reson Med 58:871–879, 2007.

Characterization of the Archaeal Thermophile Sulfolobus Turreted Icosahedral Virus Validates an Evolutionary Link among Double-Stranded DNA Viruses from All Domains of Life

ABSTRACT Icosahedral nontailed double-stranded DNA (dsDNA) viruses are present in all three domains of life, leading to speculation about a common viral ancestor that predates the divergence of Eukarya, Bacteria, and Archaea. This suggestion is supported by the shared general architecture of this group of viruses and the common fold of their major capsid protein. However, limited information on the diversity and replication of archaeal viruses, in general, has hampered further analysis. Sulfolobus turreted icosahedral virus (STIV), isolated from a hot spring in Yellowstone National Park, was the first icosahedral virus with an archaeal host to be described. Here we present a detailed characterization of the components forming this unusual virus. Using a proteomics-based approach, we identified nine viral and two host proteins from purified STIV particles. Interestingly, one of the viral proteins originates from a reading frame lacking a consensus start site. The major capsid protein (B345) was found to be glycosylated, implying a strong similarity to proteins from other dsDNA viruses. Sequence analysis and structural predication of virion-associated viral proteins suggest that they may have roles in DNA packaging, penton formation, and protein-protein interaction. The presence of an internal lipid layer containing acidic tetraether lipids has also been confirmed. The previously presented structural models in conjunction with the protein, lipid, and carbohydrate information reported here reveal that STIV is strikingly similar to viruses associated with the Bacteria and Eukarya domains of life, further strengthening the hypothesis for a common ancestor of this group of dsDNA viruses from all domains of life.