The Morphology and Assembly of Respiratory Syncytial Virus Revealed by Cryo-Electron Tomography.

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. With repeat infections throughout life, it can also cause substantial disease in the elderly and in adults with compromised cardiac, pulmonary and immune systems. RSV is a pleomorphic enveloped RNA virus in the Pneumoviridae family. Recently, the three-dimensional (3D) structure of purified RSV particles has been elucidated, revealing three distinct morphological categories: spherical, asymmetric, and filamentous. However, the native 3D structure of RSV particles associated with or released from infected cells has yet to be investigated. In this study, we have established an optimized system for studying RSV structure by imaging RSV-infected cells on transmission electron microscopy (TEM) grids by cryo-electron tomography (cryo-ET). Our results demonstrate that RSV is filamentous across several virus strains and cell lines by cryo-ET, cryo-immuno EM, and thin section TEM techniques. The viral filament length varies from 0.5 to 12 µm and the average filament diameter is approximately 130 nm. Taking advantage of the whole cell tomography technique, we have resolved various stages of RSV assembly. Collectively, our results can facilitate the understanding of viral morphogenesis in RSV and other pleomorphic enveloped viruses.

Promotion of virus assembly and organization by the measles virus matrix protein.

Measles virus (MeV) remains a major human pathogen, but there are presently no licensed antivirals to treat MeV or other paramyxoviruses. Here, we use cryo-electron tomography (cryo-ET) to elucidate the principles governing paramyxovirus assembly in MeV-infected human cells. The three-dimensional (3D) arrangement of the MeV structural proteins including the surface glycoproteins (F and H), matrix protein (M), and the ribonucleoprotein complex (RNP) are characterized at stages of virus assembly and budding, and in released virus particles. The M protein is observed as an organized two-dimensional (2D) paracrystalline array associated with the membrane. A two-layered F-M lattice is revealed suggesting that interactions between F and M may coordinate processes essential for MeV assembly. The RNP complex remains associated with and in close proximity to the M lattice. In this model, the M lattice facilitates the well-ordered incorporation and concentration of the surface glycoproteins and the RNP at sites of virus assembly.

Engineering Globular Protein Vesicles through Tunable Self-Assembly of Recombinant Fusion Proteins.

Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self-assembly of recombinant globular fusion proteins containing leucine zippers and elastin-like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermal driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi-layered. These results provide critical information to engineer globular protein vesicles via self-assembly with desired size and membrane structure.

Peripheral myelin protein 22 alters membrane architecture.

Peripheral myelin protein 22 (PMP22) is highly expressed in myelinating Schwann cells of the peripheral nervous system. PMP22 genetic alterations cause the most common forms of Charcot-Marie-Tooth disease (CMTD), which is characterized by severe dysmyelination in the peripheral nerves. However, the functions of PMP22 in Schwann cell membranes remain unclear. We demonstrate that reconstitution of purified PMP22 into lipid vesicles results in the formation of compressed and cylindrically wrapped protein-lipid vesicles that share common organizational traits with compact myelin of peripheral nerves in vivo. The formation of these myelin-like assemblies depends on the lipid-to-PMP22 ratio, as well as on the PMP22 extracellular loops. Formation of the myelin-like assemblies is disrupted by a CMTD-causing mutation. This study provides both a biochemical assay for PMP22 function and evidence that PMP22 directly contributes to membrane organization in compact myelin.

A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation.

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization and there remains no pediatric vaccine. RSV live-attenuated vaccines (LAVs) have a history of safe testing in infants; however, achieving an effective balance of attenuation and immunogenicity has proven challenging. Here we seek to engineer an RSV LAV with enhanced immunogenicity. Genetic mapping identifies strain line 19 fusion (F) protein residues that correlate with pre-fusion antigen maintenance by ELISA and thermal stability of infectivity in live RSV. We generate a LAV candidate named OE4 which expresses line 19F and is attenuated by codon-deoptimization of non-structural (NS1 and NS2) genes, deletion of the small hydrophobic (SH) gene, codon-deoptimization of the attachment (G) gene and ablation of the secreted form of G. OE4 (RSV-A2-dNS1-dNS2-ΔSH-dGm-Gsnull-line19F) exhibits elevated pre-fusion antigen levels, thermal stability, immunogenicity, and efficacy despite heavy attenuation in the upper and lower airways of cotton rats.

Self-Assembly of an α-Helical Peptide into a Crystalline Two-Dimensional Nanoporous Framework.

Sequence-specific peptides have been demonstrated to self-assemble into structurally defined nanoscale objects including nanofibers, nanotubes, and nanosheets. The latter structures display significant promise for the construction of hybrid materials for functional devices due to their extended planar geometry. Realization of this objective necessitates the ability to control the structural features of the resultant assemblies through the peptide sequence. The design of a amphiphilic peptide, 3FD-IL, is described that comprises two repeats of a canonical 18 amino acid sequence associated with straight α-helical structures. Peptide 3FD-IL displays 3-fold screw symmetry in a helical conformation and self-assembles into nanosheets based on hexagonal packing of helices. Biophysical evidence from TEM, cryo-TEM, SAXS, AFM, and STEM measurements on the 3FD-IL nanosheets support a structural model based on a honeycomb lattice, in which the length of the peptide determines the thickness of the nanosheet and the packing of helices defines the presence of nanoscale channels that permeate the sheet. The honeycomb structure can be rationalized on the basis of geometrical packing frustration in which the channels occupy defect sites that define a periodic superlattice. The resultant 2D materials may have potential as materials for nanoscale transport and controlled release applications.

Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth.

How mitochondrial glutaminolysis contributes to redox homeostasis in cancer cells remains unclear. Here we report that the mitochondrial enzyme glutamate dehydrogenase 1 (GDH1) is commonly upregulated in human cancers. GDH1 is important for redox homeostasis in cancer cells by controlling the intracellular levels of its product alpha-ketoglutarate and subsequent metabolite fumarate. Mechanistically, fumarate binds to and activates a reactive oxygen species scavenging enzyme glutathione peroxidase 1. Targeting GDH1 by shRNA or a small molecule inhibitor R162 resulted in imbalanced redox homeostasis, leading to attenuated cancer cell proliferation and tumor growth.

Rational design of helical nanotubes from self-assembly of coiled-coil lock washers.

Design of a structurally defined helical assembly is described that involves recoding of the amino acid sequence of peptide GCN4-pAA. In solution and the crystalline state, GCN4-pAA adopts a 7-helix bundle structure that resembles a supramolecular lock washer. Structurally informed mutagenesis of the sequence of GCN4-pAA afforded peptide 7HSAP1, which undergoes self-association into a nanotube via noncovalent interactions between complementary interfaces of the coiled-coil lock-washer structures. Biophysical measurements conducted in solution and the solid state over multiple length scales of structural hierarchy are consistent with self-assembly of nanotube structures derived from 7-helix bundle subunits. The dimensions of the supramolecular assemblies are similar to those observed in the crystal structure of GCN4-pAA. Fluorescence studies of the interaction of 7HSAP1 with the solvatochromic fluorophore PRODAN indicated that the nanotubes could encapsulate shape-appropriate small molecules with high binding affinity.

A stabilized headless measles virus attachment protein stalk efficiently triggers membrane fusion.

Paramyxovirus attachment and fusion (F) envelope glycoprotein complexes mediate membrane fusion required for viral entry. The measles virus (MeV) attachment (H) protein stalk domain is thought to directly engage F for fusion promotion. However, past attempts to generate truncated, fusion-triggering-competent H-stem constructs remained fruitless. In this study, we addressed the problem by testing the hypothesis that truncated MeV H stalks may require stabilizing oligomerization tags to maintain intracellular transport competence and F-triggering activity. We engineered H-stems of different lengths with added 4-helix bundle tetramerization domains and demonstrate restored cell surface expression, efficient interaction with F, and fusion promotion activity of these constructs. The stability of the 4-helix bundle tags and the relative orientations of the helical wheels of H-stems and oligomerization tags govern the kinetics of fusion promotion, revealing a balance between H stalk conformational stability and F-triggering activity. Recombinant MeV particles expressing a bioactive H-stem construct in the place of full-length H are viable, albeit severely growth impaired. Overall, we demonstrate that the MeV H stalk represents the effector domain for MeV F triggering. Fusion promotion appears linked to the conformational flexibility of the stalk, which must be tightly regulated in viral particles to ensure efficient virus entry. While the pathways toward assembly of functional fusion complexes may differ among diverse members of the paramyxovirus family, central elements of the triggering machinery emerge as highly conserved.

Receptor tyrosine kinase ErbB2 translocates into mitochondria and regulates cellular metabolism.

It is well known that ErbB2, a receptor tyrosine kinase, localizes to the plasma membrane. Here we describe a novel observation that ErbB2 also localizes in mitochondria of cancer cells and patient samples. We found that ErbB2 translocates into mitochondria through association with mtHSP70. Additionally, mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complexes of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and cellular ATP levels were also decreased. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Interestingly, cancer cells with higher levels of mtErbB2 were more resistant to the ErbB2-targeting antibody trastuzumab. Our study provides a novel perspective on the metabolic regulatory function of ErbB2 and reveals that mtErbB2 has an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics.

Co-expression of HIV-1 virus-like particles and granulocyte-macrophage colony stimulating factor by GEO-D03 DNA vaccine.

Here, we report on GEO-D03, a DNA vaccine that co-expresses non-infectious HIV-1 virus-like particles (VLPs) and the human cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). The virus-like particles display the native gp160 form of the HIV-1 Envelope glycoprotein (Env) and are designed to elicit antibody against the natural form of Env on virus and virus-infected cells. The DNA-expressed HIV Gag, Pol and Env proteins also have the potential to elicit virus-specific CD4 and CD8 T cells. The purpose of the co-expressed GM-CSF is to target a cytokine that recruits, expands and differentiates macrophages and dendritic cells to the site of VLP expression. The GEO-D03 DNA vaccine is currently entered into human trials as a prime for a recombinant modified vaccinia Ankara (MVA) boost. In preclinical studies in macaques using an SIV prototype vaccine, this vaccination regimen elicited both anti-viral T cells and antibody, and provided 70% protection against acquisition during 12 weekly rectal exposures with a heterologous SIV. Higher avidity of the Env-specific Ab for the native form of the Env in the challenge virus correlated with lower likelihood of SIV infection.

3D visualization of HIV virions by cryoelectron tomography.

The structure of the human immunodeficiency virus (HIV) and some of its components have been difficult to study in three-dimensions (3D) primarily because of their intrinsic structural variability. Recent advances in cryoelectron tomography (cryo-ET) have provided a new approach for determining the 3D structures of the intact virus, the HIV capsid, and the envelope glycoproteins located on the viral surface. A number of cryo-ET procedures related to specimen preservation, data collection, and image processing are presented in this chapter. The techniques described herein are well suited for determining the ultrastructure of bacterial and viral pathogens and their associated molecular machines in situ at nanometer resolution.

Radiation dose reduction and image enhancement in biological imaging through equally-sloped tomography.

Electron tomography is currently the highest resolution imaging modality available to study the 3D structures of pleomorphic macromolecular assemblies, viruses, organelles and cells. Unfortunately, the resolution is currently limited to 3-5nm by several factors including the dose tolerance of biological specimens and the inaccessibility of certain tilt angles. Here we report the first experimental demonstration of equally-sloped tomography (EST) to alleviate these problems. As a proof of principle, we applied EST to reconstructing frozen-hydrated keyhole limpet hemocyanin molecules from a tilt-series taken with constant slope increments. In comparison with weighted back-projection (WBP), the algebraic reconstruction technique (ART) and the simultaneous algebraic reconstruction technique (SART), EST reconstructions exhibited higher contrast, less peripheral noise, more easily detectable molecular boundaries and reduced missing wedge effects. More importantly, EST reconstructions including only two-thirds the original images appeared to have the same resolution as full WBP reconstructions, suggesting that EST can either reduce the dose required to reach a given resolution or allow higher resolutions to be achieved with a given dose. EST was also applied to reconstructing a frozen-hydrated bacterial cell from a tilt-series taken with constant angular increments. The results confirmed similar benefits when standard tilts are utilized.

Electron cryotomography of immature HIV-1 virions reveals the structure of the CA and SP1 Gag shells.

The major structural elements of retroviruses are contained in a single polyprotein, Gag, which in human immunodeficiency virus type 1 (HIV-1) comprises the MA, CA, spacer peptide 1 (SP1), NC, SP2, and p6 polypeptides. In the immature HIV-1 virion, the domains of Gag are arranged radially with the N-terminal MA domain at the membrane and C-terminal NC-SP2-p6 region nearest to the center. Here, we report the three-dimensional structures of individual immature HIV-1 virions, as obtained by electron cryotomography. The concentric shells of the Gag polyprotein are clearly visible, and radial projections of the different Gag layers reveal patches of hexagonal order within the CA and SP1 shells. Averaging well-ordered unit cells leads to a model in which each CA hexamer is stabilized by a bundle of six SP1 helices. This model suggests why the SP1 spacer is essential for assembly of the Gag lattice and how cleavage between SP1 and CA acts as a structural switch controlling maturation.

Self-assembly of block copolymers derived from elastin-mimetic polypeptide sequences.

Protein polymers derived from elastin-mimetic peptide sequences can be synthesized with near-absolute control of macromolecular architecture using genetic engineering techniques. Elastin-mimetic diblock and triblock copolymers have been prepared using this approach in which the individual elastin blocks display different phase behavior in aqueous solution. The selective collapse of the more hydrophobic blocks above the lower critical solution temperature was employed to drive the thermo-reversible self-assembly of elastin-mimetic diblock and triblock copolymer into protein-based nanoparticles and nano-textured hydrogels, respectively. These materials display considerable promise as biomaterials for applications in drug delivery and soft tissue augmentation.