Structure-first identification of RNA elements that regulate dengue virus genome architecture and replication.

The genomes of RNA viruses encode the information required for replication in host cells both in their linear sequence and in complex higher-order structures. A subset of these RNA genome structures show clear sequence conservation, and have been extensively described for well-characterized viruses. However, the extent to which viral RNA genomes contain functional structural elements-unable to be detected by sequence alone-that nonetheless are critical to viral fitness is largely unknown. Here, we devise a structure-first experimental strategy and use it to identify 22 structure-similar motifs across the coding sequences of the RNA genomes for the four dengue virus serotypes. At least 10 of these motifs modulate viral fitness, revealing a significant unnoticed extent of RNA structure-mediated regulation within viral coding sequences. These viral RNA structures promote a compact global genome architecture, interact with proteins, and regulate the viral replication cycle. These motifs are also thus constrained at the levels of both RNA structure and protein sequence and are potential resistance-refractory targets for antivirals and live-attenuated vaccines. Structure-first identification of conserved RNA structure enables efficient discovery of pervasive RNA-mediated regulation in viral genomes and, likely, other cellular RNAs.

Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures.

Nucleocapsid protein (N-protein) is required for multiple steps in betacoronaviruses replication. SARS-CoV-2-N-protein condenses with specific viral RNAs at particular temperatures making it a powerful model for deciphering RNA sequence specificity in condensates. We identify two separate and distinct double-stranded, RNA motifs (dsRNA stickers) that promote N-protein condensation. These dsRNA stickers are separately recognized by N-protein's two RNA binding domains (RBDs). RBD1 prefers structured RNA with sequences like the transcription-regulatory sequence (TRS). RBD2 prefers long stretches of dsRNA, independent of sequence. Thus, the two N-protein RBDs interact with distinct dsRNA stickers, and these interactions impart specific droplet physical properties that could support varied viral functions. Specifically, we find that addition of dsRNA lowers the condensation temperature dependent on RBD2 interactions and tunes translational repression. In contrast RBD1 sites are sequences critical for sub-genomic (sg) RNA generation and promote gRNA compression. The density of RBD1 binding motifs in proximity to TRS-L/B sequences is associated with levels of sub-genomic RNA generation. The switch to packaging is likely mediated by RBD1 interactions which generate particles that recapitulate the packaging unit of the virion. Thus, SARS-CoV-2 can achieve biochemical complexity, performing multiple functions in the same cytoplasm, with minimal protein components based on utilizing multiple distinct RNA motifs that control N-protein interactions.

Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures.

Betacoronavirus SARS-CoV-2 infections caused the global Covid-19 pandemic. The nucleocapsid protein (N-protein) is required for multiple steps in the betacoronavirus replication cycle. SARS-CoV-2-N-protein is known to undergo liquid-liquid phase separation (LLPS) with specific RNAs at particular temperatures to form condensates. We show that N-protein recognizes at least two separate and distinct RNA motifs, both of which require double-stranded RNA (dsRNA) for LLPS. These motifs are separately recognized by N-protein's two RNA binding domains (RBDs). Addition of dsRNA accelerates and modifies N-protein LLPS in vitro and in cells and controls the temperature condensates form. The abundance of dsRNA tunes N-protein-mediated translational repression and may confer a switch from translation to genome packaging. Thus, N-protein's two RBDs interact with separate dsRNA motifs, and these interactions impart distinct droplet properties that can support multiple viral functions. These experiments demonstrate a paradigm of how RNA structure can control the properties of biomolecular condensates.

Genomic RNA Elements Drive Phase Separation of the SARS-CoV-2 Nucleocapsid.

We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with viral RNA. N-protein condenses with specific RNA genomic elements under physiological buffer conditions and condensation is enhanced at human body temperatures (33°C and 37°C) and reduced at room temperature (22°C). RNA sequence and structure in specific genomic regions regulate N-protein condensation while other genomic regions promote condensate dissolution, potentially preventing aggregation of the large genome. At low concentrations, N-protein preferentially crosslinks to specific regions characterized by single-stranded RNA flanked by structured elements and these features specify the location, number, and strength of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is RNA sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules, and therefore presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2.

Physical and Functional Analysis of Viral RNA Genomes by SHAPE.

RNA viruses encode the information required to usurp cellular metabolism and gene regulation and to enable their own replication in two ways: in the linear sequence of their RNA genomes and in higher-order structures that form when the genomic RNA strand folds back on itself. Application of high-resolution SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) structure probing to viral RNA genomes has identified numerous new regulatory elements, defined new principles by which viral RNAs interact with the cellular host and evade host immune responses, and revealed relationships between virus evolution and RNA structure. This review summarizes our current understanding of genome structure-function interrelationships for RNA viruses, as informed by SHAPE structure probing, and outlines opportunities for future studies.

Pervasive tertiary structure in the dengue virus RNA genome.

RNA virus genomes are efficient and compact carriers of biological information, encoding information required for replication both in their primary sequences and in higher-order RNA structures. However, the ubiquity of RNA elements with higher-order folds-in which helices pack together to form complex 3D structures-and the extent to which these elements affect viral fitness are largely unknown. Here we used single-molecule correlated chemical probing to define secondary and tertiary structures across the RNA genome of dengue virus serotype 2 (DENV2). Higher-order RNA structures are pervasive and involve more than one-third of nucleotides in the DENV2 genomic RNA. These 3D structures promote a compact overall architecture and contribute to viral fitness. Disrupting RNA regions with higher-order structures leads to stable, nonreverting mutants and could guide the development of vaccines based on attenuated RNA viruses. The existence of extensive regions of functional RNA elements with tertiary folds in viral RNAs, and likely many other messenger and noncoding RNAs, means that there are significant regions with pocket-containing surfaces that may serve as novel RNA-directed drug targets.

Design and Crystallography of Self-Assembling RNA Nanostructures.

Biological RNA architectures are composed of autonomously folding modules which can be tailored as building blocks for the construction of RNA nanostructures. Designed base pair interactions allow complex nano-objects to self-assemble from simple RNA motifs. X-ray crystallography plays an important role in both the design and analysis of such RNA nanostructures. Here, we describe methods for the design and X-ray crystallographic structure analysis of an RNA square and two different triangles, which self-assemble from short oligonucleotides and serve as a platform for building functional nano-sized nucleic acid architectures.

Structure of the Ribosomal RNA Decoding Site Containing a Selenium-Modified Responsive Fluorescent Ribonucleoside Probe.

Comprehensive understanding of the structure-function relationship of RNA both in real time and at atomic level will have a profound impact in advancing our understanding of RNA functions in biology. Here, we describe the first example of a multifunctional nucleoside probe, containing a conformation-sensitive fluorophore and an anomalous X-ray diffraction label (5-selenophene uracil), which enables the correlation of RNA conformation and recognition under equilibrium and in 3D. The probe incorporated into the bacterial ribosomal RNA decoding site, fluorescently reports antibiotic binding and provides diffraction information in determining the structure without distorting native RNA fold. Further, by comparing solution binding data and crystal structure, we gained insight on how the probe senses ligand-induced conformational change in RNA. Taken together, our nucleoside probe represents a new class of biophysical tool that would complement available tools for functional RNA investigations.

Ligand Optimization by Improving Shape Complementarity at a Hepatitis C Virus RNA Target.

Crystal structure analysis revealed key interactions of a 2-amino-benzimidazole viral translation inhibitor that captures an elongated conformation of an RNA switch target in the internal ribosome entry site (IRES) of hepatitis C virus (HCV). Here, we have designed and synthesized quinazoline derivatives with improved shape complementarity at the ligand binding site of the viral RNA target. A spiro-cyclopropyl modification aimed at filling a pocket in the back of the RNA binding site led to a 5-fold increase of ligand affinity while a slightly more voluminous dimethyl substitution at the same position did not improve binding. We demonstrate that precise shape complementarity based solely on hydrophobic interactions contributes significantly to ligand binding even at a hydrophilic RNA target site such as the HCV IRES conformational switch.

Crystal-Structure-Guided Design of Self-Assembling RNA Nanotriangles.

RNA nanotechnology uses RNA structural motifs to build nanosized architectures that assemble through selective base-pair interactions. Herein, we report the crystal-structure-guided design of highly stable RNA nanotriangles that self-assemble cooperatively from short oligonucleotides. The crystal structure of an 81 nucleotide nanotriangle determined at 2.6 Šresolution reveals the so-far smallest circularly closed nanoobject made entirely of double-stranded RNA. The assembly of the nanotriangle architecture involved RNA corner motifs that were derived from ligand-responsive RNA switches, which offer the opportunity to control self-assembly and dissociation.

Conformational flexibility of viral RNA switches studied by FRET.

The function of RNA switches involved in the regulation of transcription and translation relies on their ability to adopt different, structurally well-defined states. A new class of ligand-responsive RNA switches, which we recently discovered in positive strand RNA viruses, are distinct from conventional riboswitches. The viral switches undergo large conformational changes in response to ligand binding while retaining the same secondary structure in their free and ligand-bound forms. Here, we describe FRET experiments to study folding and ligand binding of the viral RNA switches. In addition to reviewing previous approaches involving RNA model constructs which were directly conjugated with fluorescent dyes, we outline the design and application of new modular constructs for FRET experiments, in which dye labeling is achieved by hybridization of a core RNA switch module with universal DNA fluorescent probes. As an example, folding and ligand binding of the RNA switch from the internal ribosome entry site of hepatitis C virus is studied comparatively with conventional and modular FRET constructs.

Ligand-responsive RNA mechanical switches.

Ligand-responsive RNA mechanical switches represent a new class of simple switching modules that adopt well-defined ligand-free and bound conformational states, distinguishing them from metabolite-sensing riboswitches. Initially discovered in the internal ribosome entry site (IRES) of hepatitis C virus (HCV), these RNA switch motifs were found in the genome of diverse other viruses. Although large variations are seen in sequence and local secondary structure of the switches, their function in viral translation initiation that requires selective ligand recognition is conserved. We recently determined the crystal structure of an RNA switch from Seneca Valley virus (SVV) which is able to functionally replace the switch of HCV. The switches from both viruses recognize identical cognate ligands despite their sequence dissimilarity. Here, we describe the discovery of 7 new switches in addition to the previously established 5 examples. We highlight structural and functional features unique to this class of ligand-responsive RNA mechanical switches and discuss implications for therapeutic development and the construction of RNA nanostructures.

Functional conservation despite structural divergence in ligand-responsive RNA switches.

An internal ribosome entry site (IRES) initiates protein synthesis in RNA viruses, including the hepatitis C virus (HCV). We have discovered ligand-responsive conformational switches in viral IRES elements. Modular RNA motifs of greatly distinct sequence and local secondary structure have been found to serve as functionally conserved switches involved in viral IRES-driven translation and may be captured by identical cognate ligands. The RNA motifs described here constitute a new paradigm for ligand-captured switches that differ from metabolite-sensing riboswitches with regard to their small size, as well as the intrinsic stability and structural definition of the constitutive conformational states. These viral RNA modules represent the simplest form of ligand-responsive mechanical switches in nucleic acids.

2-Aminobenzoxazole ligands of the hepatitis C virus internal ribosome entry site.

2-Aminobenzoxazoles have been synthesized as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The compounds were designed to explore the less basic benzoxazole system as a replacement for the core scaffold in previously discovered benzimidazole viral translation inhibitors. Structure-activity relationships in the target binding of substituted benzoxazole ligands were investigated.

Aryl-substituted aminobenzimidazoles targeting the hepatitis C virus internal ribosome entry site.

We describe the exploration of N1-aryl-substituted benzimidazoles as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The design of the compounds was guided by the co-crystal structure of a benzimidazole viral translation inhibitor in complex with the RNA target. Structure-binding activity relationships of aryl-substituted benzimidazole ligands were established that were consistent with the crystal structure of the translation inhibitor complex.

Hepatitis C virus translation inhibitors targeting the internal ribosomal entry site.

The internal ribosome entry site (IRES) in the 5' untranslated region (UTR) of the hepatitis C virus (HCV) genome initiates translation of the viral polyprotein precursor. The unique structure and high sequence conservation of the 5' UTR render the IRES RNA a potential target for the development of selective viral translation inhibitors. Here, we provide an overview of approaches to block HCV IRES function by nucleic acid, peptide, and small molecule ligands. Emphasis will be given to the IRES subdomain IIa, which currently is the most advanced target for small molecule inhibitors of HCV translation. The subdomain IIa behaves as an RNA conformational switch. Selective ligands act as translation inhibitors by locking the conformation of the RNA switch. We review synthetic procedures for inhibitors as well as structural and functional studies of the subdomain IIa target and its ligand complexes.