Structural definition of substrate recognition by model RNA capping enzymes and the identification of a novel class of viral RNA capping enzymes

The RNA cap structure is a fundamental feature of most known eukaryotic mRNAs and some viral RNAs, It is important for the stability, transport and translation of mRNAs. It is co-transcriptionally synthesized via the action of 3 consecutive enzymatic reactions: (1) A RNA triphosphatase which cleaves off the 5' terminal phosphate of nascent RNAs; (2) A RNA guanylyltransferase which transfers a GMP moiety onto the acceptor RNA; (3) A RNA (guanine-N7) methyltransferase which methylates the cap guanine at the N7 position. Through the end of the 1990's until now, the crystal structures of several capping enzymes have been solved. However, these structures, although very insightful in themselves, failed to provide any instructive information on several key issues regarding enzyme-substrate interactions. For instance, one of the first breakthrough crystallographic studies in RNA capping chemistry led to the elucidation of the yeast RNA triphosphatase structure (the Cet1 protein). However, in the crystal structure, the Cet1 protein was bound to a sulphate molecule, which was hypothesised to be mimicking the product of the RNA triphosphatase reaction- a phosphate molecule.The inability to capture the RNA triphosphatase in complex with its ligands is probably on account of the inherent thermodynamic instability of this protein when bound to RNA or a nucleotide. A structural definition of the active site of the yeast RNA triphosphatase in complex with an appropriate substrate is still lacking. In addition, the elucidation of the structure of the RNA guanylyltransferase of the Paramecium bursaria chlorella virus -1 (PBCV-1) in several different conformations has been a landmark study which greatly contributed towards the understanding of the catalytic pathway of this model enzyme. On the other hand, despite the presence of the natural substrate-GTP, within the active site of the enzyme, the rationale behind the GTP specificity of RNA guanylyltransferase remains poorly understood. Moreover, a molecular mechanism for the RNA guanylyltransferase reaction is still missing. Finally, the importance of the RNA cap for the process of eukaryotic translation is undisputable. However, the relationship between the RNA cap and translation has been mostly studied indirectly through proteins which bind to the cap structure. Most studies pertaining directly to the impact of the binding of the RNA cap structure have been restricted to investigating the inhibitory potential of various cap analogues on the translation process. Studies on the effects of modified RNA caps at the 5' ends of RNAs have only started in the last few years, and more importantly, the necessity of the N7-methyl group on RNA cap analogues had not been addressed. This thesis therefore aims to provide a structural insight into the structural dynamics of enzyme-ligand(s) interactions of the model S. cerevisiae's RNA triphosphatase and the PBCV-1 RNA guanylyltransferase. In addition, we show that purine analogues can be a useful tool for the study of several cellular processes, such as RNA translation. In the process we have uncovered a novel class of RNA capping enzyme in the flavivirus genus of the Flaviviridae family of RNA viruses, thus providing a more succinct insight into the flaviviral replication complex

Deciphering the molecular basis for nucleotide selection by the West Nile virus RNA helicase

The West Nile virus RNA helicase uses the energy derived from the hydrolysis of nucleotides to separate complementary strands of RNA. Although this enzyme has a preference for ATP, the bias towards this purine nucleotide cannot be explained on the basis of specific protein–ATP interactions. Moreover, the enzyme does not harbor the characteristic Q-motif found in other helicases that regulates binding to ATP. In the present study, we used structural homology modeling to generate a model of the West Nile virus RNA helicase active site that provides instructive findings on the interaction between specific amino acids and the ATP substrate. In addition, we evaluated both the phosphohydrolysis and the inhibitory potential of a collection of 30 synthetic purine analogs. A structure-guided alanine scan of 16 different amino acids was also performed to clarify the contacts that are made between the enzyme and ATP. Our study provides a molecular rationale for the bias of the enzyme for ATP by highlighting the specific functional groups on ATP that are important for binding. Moreover, we identified three new essential amino acids (Arg-185, Arg-202 and Asn-417) that are critical for phosphohydrolysis. Finally, we provide evidence that a region located upstream of motif I, which we termed the nucleotide specificity region, plays a functional role in nucleotide selection which is reminiscent to the role exerted by the Q-motif found in other helicases

Resistance Patterns Associated with HCV NS5A Inhibitors Provide Limited Insight into Drug Binding

Direct-acting antivirals (DAAs) have significantly improved the treatment of infection with the hepatitis C virus. A promising class of novel antiviral agents targets the HCV NS5A protein. The high potency and broad genotypic coverage are favorable properties. NS5A inhibitors are currently assessed in advanced clinical trials in combination with viral polymerase inhibitors and/or viral protease inhibitors. However, the clinical use of NS5A inhibitors is also associated with new challenges. HCV variants with decreased susceptibility to these drugs can emerge and compromise therapy. In this review, we discuss resistance patterns in NS5A with focus prevalence and implications for inhibitor binding

Enzymatic Synthesis of RNAs Capped with Nucleotide Analogues Reveals the Molecular Basis for Substrate Selectivity of RNA Capping Enzyme: Impacts on RNA Metabolism

<div><p>RNA cap binding proteins have evolved to specifically bind to the N7-methyl guanosine cap structure found at the 5’ ends of eukaryotic mRNAs. The specificity of RNA capping enzymes towards GTP for the synthesis of this structure is therefore crucial for mRNA metabolism. The fact that ribavirin triphosphate was described as a substrate of a viral RNA capping enzyme, raised the possibility that RNAs capped with nucleotide analogues could be generated <i>in cellulo</i>. Owing to the fact that this prospect potentially has wide pharmacological implications, we decided to investigate whether the active site of the model <i>Paramecium</i><i>bursaria</i><i> Chlorella virus-1</i> RNA capping enzyme was flexible enough to accommodate various purine analogues. Using this approach, we identified several key structural determinants at each step of the RNA capping reaction and generated RNAs harboring various different cap analogues. Moreover, we monitored the binding affinity of these novel capped RNAs to the eIF4E protein and evaluated their translational properties <i>in cellulo</i>. Overall, this study establishes a molecular rationale for the specific selection of GTP over other NTPs by RNA capping enzyme It also demonstrates that RNAs can be enzymatically capped with certain purine nucleotide analogs, and it also describes the impacts of modified RNA caps on specific steps involved in mRNA metabolism. For instance, our results indicate that the N7-methyl group of the classical N7-methyl guanosine cap is not always indispensable for binding to eIF4E and subsequently for translation when compensatory modifications are present on the capped residue. Overall, these findings have important implications for our understanding of the molecular determinants involved in both RNA capping and RNA metabolism.</p> </div

<i>In</i><i>cellulo</i> and <i>in</i><i>vitro</i> properties of the novel cap analogues

<p>(A) Schematic representation of the experimental procedure for the determination of the translation efficiency of differentially capped lucA₆₀ RNA in HEK293 cells. (B) The relative translation efficiency was experimentally determined by quantifying <i>firefly</i> luciferase activity relative to the amount of total protein 6 hr post-transfection. Experimental data was adjusted relative to the capping efficiency (as determined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075310#pone-0075310-t001" target="_blank">Table 1</a>) of each analogue, and rationalized onto the ^m7G cap. The error associated with each data set is less than ± 0.1. (*) indicates more than 1.5 fold difference relative to the translation efficiency of a naturally capped RNA. (C) The relative RNA level was evaluated by quantifying the amount of lucA60 RNA relative to the GAPDH RNA by qRT-PCR 0 hr and 6 hr post-transfection. (D) Binding to eIF4E was determined by fluorescence spectroscopy with a 30 nt long differentially capped RNA molecule. (*) indicates more than 1.5 fold difference relative to the binding observed for the natural ^m7G capped RNA.</p

The RNA capping mechanism and the nucleotide analogues tested

<p>(A) The two-step RNA guanylyltransferase reaction. (B) The GTP binding site of the PBCV-1 GTase (PDB 1CKN). Residues shown are those interacting with the base and the sugar moiety. (C) Nucleotide analogues used in this study.</p

<i>In</i><i>vitro</i> characterization of 3’ O-methyl GTP (A₂₂) as an inhibitor of translation and as a binding partner to eIF4E

<p>(A) To the rabbit reticulocyte lysate (Promega) <i>in </i><i>vitro</i> translation system, ^m7G capped lucA₆₀ RNA (1µg) and increasing amounts of NTP was added. Luciferase activity was measured after 10 minutes and plotted relative to the activity in the absence of any nucleotides. (B) Increasing amounts of NTP were added to a 2 µM solution of the purified protein in a binding buffer (50 mM Tris/HCl, pH 8.0, and 50 mM KOAc) and following excitation of tryptophan residues at 290 nm the emission spectrum was scanned from 310 to 440 nm. A saturation isotherm was generated from these data by plotting the change in fluorescence intensity at 333 nm as a function of added NTP. The data was fit to a binding curve. (■) indicates ^m7GTP; (▲) indicates 3’ O methyl GTP (A₂₂); and (●) represents GTP.</p

The flavivirus NS5 protein is a true RNA guanylyltransferase that catalyzes a two-step reaction to form the RNA cap structure

The 5′-end of the flavivirus genome harbors a methylated m7GpppA2′OMe cap structure, which is generated by the virus-encoded RNA triphosphatase, RNA (guanine-N7) methyltransferase, nucleoside 2′-O-methyltransferase, and RNA guanylyltransferase. The presence of the flavivirus guanylyltransferase activity in NS5 has been suggested by several groups but has not been empirically proven. Here we provide evidence that the N-terminus of the flavivirus NS5 protein is a true RNA guanylyltransferase. We demonstrate that GTP can be used as a substrate by the enzyme to form a covalent GMP–enzyme intermediate via a phosphoamide bond. Mutational studies also confirm the importance of a specific lysine residue in the GTP binding site for the enzymatic activity. We show that the GMP moiety can be transferred to the diphosphate end of an RNA transcript harboring an adenosine as the initiating residue. We also demonstrate that the flavivirus RNA triphosphatase (NS3 protein) stimulates the RNA guanylyltransferase activity of the NS5 protein. Finally, we show that both enzymes are sufficient and necessary to catalyze the de novo formation of a methylated RNA cap structure in vitro using a triphosphorylated RNA transcript. Our study provides biochemical evidence that flaviviruses encode a complete RNA capping machinery

Interactions of the Disordered Domain II of Hepatitis C Virus NS5A with Cyclophilin A, NS5B, and Viral RNA Show Extensive Overlap

Domain II of the nonstructural protein 5 (NS5A) of the hepatitis C virus (HCV) is involved in intermolecular interactions with the viral RNA genome, the RNA-dependent RNA polymerase NS5B, and the host factor cyclophilin A (CypA). However, domain II of NS5A (NS5A^DII) is largely disordered, which makes it difficult to characterize the protein–protein or protein–nucleic acid interfaces. Here we utilized a mass spectrometry-based protein footprinting approach in attempts to characterize regions forming contacts between NS5A^DII and its binding partners. In particular, we compared surface topologies of lysine and arginine residues in the context of free and bound NS5A^DII. These experiments have led to the identification of an RNA binding motif (³⁰⁵RSR­KFPR³¹¹) in an arginine-rich region of NS5A^DII. Furthermore, we show that K308 is indispensable for both RNA and NS5B binding, whereas W316, further downstream, is essential for protein–protein interactions with CypA and NS5B. Most importantly, NS5A^DII binding to NS5B involves a region associated with RNA binding within NS5B. This interaction down-regulated RNA synthesis by NS5B, suggesting that NS5A^DII modulates the activity of NS5B and potentially regulates HCV replication