Eric Alonas

Human Respiratory Syncytial Virus Nucleoprotein and Inclusion Bodies Antagonize the Innate Immune Response Mediated by MDA5 and MAVS

ABSTRACT Currently, the spatial distribution of human respiratory syncytial virus (hRSV) proteins and RNAs in infected cells is still under investigation, with many unanswered questions regarding the interaction of virus-induced structures and the innate immune system. Very few studies of hRSV have used subcellular imaging as a means to explore the changes in localization of retinoic-acid-inducible gene-I (RIG-I)-like receptors or the mitochondrial antiviral signaling (MAVS) protein, in response to the infection and formation of viral structures. In this investigation, we found that both RIG-I and melanoma differentiation-associated gene 5 (MDA5) colocalized with viral genomic RNA and the nucleoprotein (N) as early as 6 h postinfection (hpi). By 12 hpi, MDA5 and MAVS were observed within large viral inclusion bodies (IB). We used a proximity ligation assay (PLA) and determined that the N protein was in close proximity to MDA5 and MAVS in IBs throughout the course of the infection. Similar results were found with the transient coexpression of N and the phosphoprotein (P). Additionally, we demonstrated that the localization of MDA5 and MAVS in IBs inhibited the expression of interferon β mRNA 27-fold following Newcastle disease virus infection. From these data, we concluded that the N likely interacts with MDA5, is in close proximity to MAVS, and localizes these molecules within IBs in order to attenuate the interferon response. To our knowledge, this is the first report of a specific function for hRSV IBs and of the hRSV N protein as a modulator of the innate immune response.

Quantifying RNA–protein interactions in situ using modified-MTRIPs and proximity ligation

The stabilization, translation and degradation of RNA are regulated by interactions between trans-acting factors, such as microRNA and RNA-binding proteins (RBP). In order to investigate the relationships between these events and their significance, a method that detects the localization of these interactions within a single cell, as well as their variability across a cell population, is needed. To visualize and quantify RNA–protein interactions in situ, we developed a proximity ligation assay (PLA) that combined peptide-modified, multiply-labelled tetravalent RNA imaging probes (MTRIPs), targeted to sequences near RBP binding sites, with proximity ligation and rolling circle amplification (RCA). Using this method, we detected and quantified, with single-interaction sensitivity, the localization and frequency of interactions of the human respiratory syncytial virus (hRSV) nucleocapsid protein (N) with viral genomic RNA (gRNA). We also described the effects of actinomycin D (actD) on the interactions of HuR with β-actin mRNA and with poly(A)+ mRNA at both native and increased HuR expression levels.

Structural Analysis of Respiratory Syncytial Virus Reveals the Position of M2-1 between the Matrix Protein and the Ribonucleoprotein Complex

ABSTRACT Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family of nonsegmented, negative-sense, single-stranded RNA genome viruses, is a leading cause of lower respiratory tract infections in infants, young children, and the elderly or immunocompromised. There are many open questions regarding the processes that regulate human RSV (hRSV) assembly and budding. Here, using cryo-electron tomography, we identified virus particles that were spherical, filamentous, and asymmetric in structure, all within the same virus preparation. The three particle morphologies maintained a similar organization of the surface glycoproteins, matrix protein (M), M2-1, and the ribonucleoprotein (RNP). RNP filaments were traced in three dimensions (3D), and their total length was calculated. The measurements revealed the inclusion of multiple full-length genome copies per particle. RNP was associated with the membrane whenever the M layer was present. The amount of M coverage ranged from 24% to 86% in the different morphologies. Using fluorescence light microscopy (fLM), direct stochastic optical reconstruction microscopy (dSTORM), and a proximity ligation assay (PLA), we provide evidence illustrating that M2-1 is located between RNP and M in isolated viral particles. In addition, regular spacing of the M2-1 densities was resolved when hRSV viruses were imaged using Zernike phase contrast (ZPC) cryo-electron tomography. Our studies provide a more complete characterization of the hRSV virion structure and substantiation that M and M2-1 regulate virus organization. IMPORTANCE hRSV is a leading cause of lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. We used cryo-electron tomography and Zernike phase contrast cryo-electron tomography to visualize populations of purified hRSV in 3D. We observed the three distinct morphologies, spherical, filamentous, and asymmetric, which maintained comparable organizational profiles. Depending on the virus morphology examined, the amount of M ranged from 24% to 86%. We complemented the cryo-imaging studies with fluorescence microscopy, dSTORM, and a proximity ligation assay to provide additional evidence that M2-1 is incorporated into viral particles and is positioned between M and RNP. The results highlight the impact of M and M2-1 on the regulation of hRSV organization.

Achieving λ/10 resolution CW STED nanoscopy with a Ti:Sapphire oscillator.

In this report, a Ti:Sapphire oscillator was utilized to realize synchronization-free stimulated emission depletion (STED) microscopy. With pump power of 4.6 W and sample irradiance of 310 mW, we achieved super-resolution as high as 71 nm. With synchronization-free STED, we imaged 200 nm nanospheres as well as all three cytoskeletal elements (microtubules, intermediate filaments, and actin filaments), clearly demonstrating the resolving power of synchronization-free STED over conventional diffraction limited imaging. It also allowed us to discover that, Dylight 650, exhibits improved performance over ATTO647N, a fluorophore frequently used in STED. Furthermore, we applied synchronization-free STED to image fluorescently-labeled intracellular viral RNA granules, which otherwise cannot be differentiated by confocal microscopy. Thanks to the widely available Ti:Sapphire oscillators in multiphoton imaging system, this work suggests easier access to setup super-resolution microscope via the synchronization-free STED.

Mirror-enhanced super-resolution microscopy

Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of ~100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes. Axial sectioning can be obtained with wavelength modulation or by controlling the spacer between the mirror and the specimen. With no additional complexity, the mirror-assisted excitation confinement enhanced the axial resolution six-fold and the lateral resolution two-fold for STED, which together achieved 19-nm resolution to resolve the inner rim of a nuclear pore complex and to discriminate the contents of 120 nm viral filaments. The ability to increase the lateral resolution and decrease the thickness of an axial section using mirror-enhanced STED without increasing the laser power is of great importance for imaging biological specimens, which cannot tolerate high laser power.

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5–15 d for an individual experienced in cryo-EM.

A cytokine immunosensor for Multiple Sclerosis detection based upon label-free electrochemical impedance spectroscopy using electroplated printed circuit board electrodes.

A biosensor for the serum cytokine, Interleukin-12 (IL-12), based upon a label-free electrochemical impedance spectroscopy (EIS) monitoring approach is described. Overexpression of IL-12 has been correlated to the diagnosis of Multiple Sclerosis (MS). An immunosensor has been fabricated by electroplating gold onto a disposable printed circuit board (PCB) electrode and immobilizing anti-IL-12 monoclonal antibodies (MAb) onto the surface of the electrode. This approach yields a robust sensor that facilitates reproducible mass fabrication and easy alteration of the electrode shape. Results indicate that this novel PCB sensor can detect IL-12 at physiological levels, <100 fM with f-values of 0.05 (typically <0.0001) in a label-free and rapid manner. A linear (with respect to log concentration) detectable range was achieved. Detection in a complex biological solution is also explored; however, significant loss of dynamic range is noted in the 100% complex solution. The cost effective approach described here can be used potentially for diagnosis of diseases (like MS) with known biomarkers in body fluids and for monitoring physiological levels of biomolecules with healthcare, food, and environmental relevance.

Probes for Intracellular RNA Imaging in Live Cells

RNA localization, dynamics, and regulation are becoming increasingly important to our basic understanding of gene expression and RNA virus pathogenesis. An improved understanding of these processes will be necessary in order to identify new drug targets, as well as to create models of gene expression networks. Much of this new understanding will likely come from imaging studies of RNA, which can generate the spatiotemporal information necessary to characterize RNA within the cellular milieu. Ideally, this would be performed imaging native, nonengineered RNAs, but the approaches for performing these experiments are still evolving. In order for them to reach their potential, it is critical that they have characteristics that allow for the tracking of RNA throughout their life cycle. This chapter presents an overview of RNA imaging methodologies, and focuses on a single RNA sensitive method, employing exogenous probes, for imaging, native, nonengineered RNA in live cells.

A Low-Cost Electrochemical Biosensor for Rapid Bacterial Detection

A three-electrode electrochemical biosensor has been developed using printed circuit boards for detecting bacterial contamination. This low-cost, small-size device consists of thin-film gold electrodes and is fabricated using photo-lithography paired with electro-deposition. Pathogen presence is deduced by detection of change in electrical impedance caused by binding of the pathogen to bio-receptors coated on the sensor surface. The biosensor has a total area of 3.2 cm and requires only 100 L of test sample for detection. The sensor geometry has been optimized using techniques from Design of Experiments, and the device can be operated using a small ac excitation potential of magnitude 5 mV. The sensor is tested on the common food-borne pathogen Salmonella typhimurium and is able detect bacterial concentrations of the order of 500 CFU/mL within 6 min. In this paper, the design and fabrication of the biosensor is detailed along with the experiments that validate its performance.

High-throughput templated multisegment synthesis of gold nanowires and nanorods.

A cost-effective, high-throughput method for generating gold nanowires and/or nanorods based on a multisegment template electrodeposition approach is described. Using this method, multiple nanowires/nanorods can be generated from a single pore of alumina template membranes by alternately depositing segments of desirable (e.g., gold) and non-desirable metals (e.g., silver), followed by dissolution of the template and the non-desirable metal. Critical cost analysis indicates substantial savings in material requirements, processing times, and processing costs compared to the commonly used single-segment method. In addition to solid gold nanowires/nanorods, high yields of porous gold nanowires/nanorods are obtained by depositing alternate segments of gold-silver alloy and silver from the same gold-silver plating solution followed by selective dissolution of the silver from both segments. It is anticipated that this high-throughput method for synthesizing solid and porous gold nanowires and nanorods will accelerate their use in sensing, electronic, and biomedical applications.