Visualizing the Determinants of Viral RNA Recognition by Innate Immune Sensor RIG-I

SummaryRetinoic acid inducible gene-I (RIG-I) is a key intracellular immune receptor for pathogenic RNAs, particularly from RNA viruses. Here, we report the crystal structure of human RIG-I bound to a 5′ triphosphorylated RNA hairpin and ADP nucleotide at 2.8 Å resolution. The RNA ligand contains all structural features that are essential for optimal recognition by RIG-I, as it mimics the panhandle-like signatures within the genome of negative-stranded RNA viruses. RIG-I adopts an intermediate, semiclosed conformation in this product state of ATP hydrolysis. The structure of this complex allows us to visualize the first steps in RIG-I recognition and activation upon viral infection

Experimental demonstration of RGB LED-based optical camera communications

Red, green, and blue (RGB) light-emitting diodes (LEDs) are widely used in everyday illumination, particularly where color-changing lighting is required. On the other hand, digital cameras with color filter arrays over image sensors have been also extensively integrated in smart devices. Therefore, optical camera communications (OCC) using RGB LEDs and color cameras is a promising candidate for cost-effective parallel visible light communications (VLC). In this paper, a single RGB LED-based OCC system utilizing a combination of undersampled phase-shift on off keying (UPSOOK), wavelength-division multiplexing (WDM), and multiple-input multiple-output (MIMO) techniques is designed, which offers higher space efficiency (3 bits/Hz/LED), long-distance, and nonflickering VLC data transmission. A proof-of-concept test bed is developed to assess the bit-error-rate performance of the proposed OCC system. The experimental results show that the proposed system using a single commercially available RGB LED and a standard 50-frame/s camera is able to achieve a data rate of 150 bits/s over a range of up to 60 m

Real-Time 262-Mb/s Visible Light Communication With Digital Predistortion Waveform Shaping

A digital predistortion waveform shaping scheme combined with a blue filter is proposed to optimize both the rise and fall times of a light-emitting diode (LED) and the optical receiver current of the signal of the real-time visible light communication (VLC) system. The proposed scheme is implemented on a field-programmable gate array (FPGA) and a digital-to-analog converter based test bed, which is flexible and reconfigurable by programming the FPGA to match different LED characteristics and varied data rates. A 262-Mb/s non-return-to-zero on-off keying modulation based real-time VLC link with a bit error rate of less than 1.0×10−6 is achieved over a transmission distance of 5.0 m, which uses a single white phosphorous LED with a limited power of 0.1 W

High-resolution HDX-MS reveals distinct mechanisms of RNA recognition and activation by RIG-I and MDA5

ABSTRACT RIG-I and MDA5 are the major intracellular immune receptors that recognize viral RNA species and undergo a series of conformational transitions leading to the activation of the interferon-mediated antiviral response. However, to date, full-length RLRs have resisted crystallographic efforts and a molecular description of their activation pathways remains hypothetical. Here we employ hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) to probe the apo states of RIG-I and MDA5 and to dissect the molecular details with respect to distinct RNA species recognition, ATP binding and hydrolysis and CARDs activation. We show that human RIG-I maintains an auto-inhibited resting state owing to the intra-molecular HEL2i-CARD2 interactions while apo MDA5 lacks the analogous intra-molecular interactions and therefore adopts an extended conformation. Our work demonstrates that RIG-I binds and responds differently to short triphosphorylated RNA and long duplex RNA and that sequential addition of RNA and ATP triggers specific allosteric effects leading to RIG-I CARDs activation. We also present a high-resolution protein surface mapping technique that refines the cooperative oligomerization model of neighboring MDA5 molecules on long duplex RNA. Taken together, our data provide a high-resolution view of RLR activation in solution and offer new evidence for the molecular mechanism of RLR activation

2-Cyanoisonicotinamide Conjugation: A Facile Approach to Generate Potent Peptide Inhibitors of the Zika Virus Protease

The rapid generation and modification of macrocyclic peptides in medicinal chemistry is an ever-growing area that can present various synthetic challenges. The reaction between N-terminal cysteine and 2-cyanoisonicotinamide is a new biocompatible click reaction that allows rapid access to macrocyclic peptides. Importantly, 2-cyanoisonicotinamide can be attached to different linkers directly during solid-phase peptide synthesis. The synthesis involves only commercially available precursors, allowing for a fully automated process. We demonstrate the approach for four cyclic peptide ligands of the Zika virus protease NS2B-NS3. Although all peptides display the substrate recognition motif, the activity strongly depends on the linker length, with the shortest cyclization linker corresponding to highest activity (K i = 0.64 μM). The most active cyclic peptide displays affinity 78 times higher than that of its linear analogue. We solved a crystal structure of the proteolytically cleaved ligand and synthesized it by applying the presented chemistry to peptide ligation.DE19010001

Comparative observations on the squamous-columnar junction of Von Ebner’s glandular duct at the bottom of vallate papillae in dogs, rats, mice and human

Background: This paper aims to comparatively observe similarities of squamous-columnar junction (SCJ) at the opening of Von Ebner's glandular ducts at the vallate papillae in dogs, mice, rats and humans, lay a foundation for the selection of the model in future study of the carcinogenesis in SCJ at vallate papillae. Materials and methods: The localization of the vallate papillae in three laboratory animals and humans was comparatively observed. The differences of SCJ at vallate papillae were comparatively observed by Alcian blue, immunohistochemistry and HE staining. Results: Anatomically, the canine vallate papillae were most similar to those of humans in location, whereas mice and rats only had a single, Ω-shaped, vallate papilla lying directly anterior to the posterior border of the intermolar eminence. In histology, the SCJ of dogs lacked a transition zone similar to that of the human SCJ, and there was glandular epithelium secreting acidic mucus at the opening of the rats’ Von Ebner's glandular ducts. All of this suggested that the histological structure of SCJ in rats and dogs is more distinct from that of humans, whereas the histological structure of SCJ at vallate papilla in mice was more similar. Conclusions: The structure of SCJ at vallate papilla in mice is most similar to that of humans, so we conclude that mouse is the most suitable model for studying tumorigenesis in SCJ at vallate papillae in these three common laboratory animals

The Hexamer Structure of the Rift Valley Fever Virus Nucleoprotein Suggests a Mechanism for its Assembly into Ribonucleoprotein Complexes

Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

The NS3 protein from dengue virus : structure and function

170 p.The Dengue virus belongs to the flavivirus genus which includes other important human pathogens such as Yellow fever, Japanese encephalitis and West Nile virus within the Flaviviridae family of enveloped viruses. Dengue virus affects several million people annually, with no effective therapy at hand. The Dengue virion contains a single stranded, positive sense (+) RNA genome of approximately 11 kb which is translated into a large polyprotein containing both structural proteins and nonstructural proteins during the infectious life cycle. The non-structural protein 3 (NS3) is a multifunctional protein of 69 kDa. The N terminal domain of NS3 is a serine protease with NS2B as a cofactor essential for viral polyprotein processing. The C terminal domain is a RNA helicase and a nucleoside 5* triphosphatase, involved in viral replication and virus particle assembly. Thus, NS3 plays an important role in viral life cycle and represents a target for the development of specific antiviral inhibitors.DOCTOR OF PHILOSOPHY (SBS