GTP and RNA cap analog competition analysis with CHIKV nsP1.

<p>GTP and RNA cap analog competition analysis with CHIKV nsP1.</p

Purified CHIKV nsP1 is enzymatically active.

<p>Purified CHIKV nsP1 was incubated with GTP-680 and either 100 μM GTP, 100 μM SAM or 100 μM SAM and GTP for 1 hour. Reactions were resolved on SDS page gels and gels were scanned for fluorescence on an Odyssey Clx Infrared Imaging System before being stained with Commassie. (A) Guanylation was quantified and normalized to protein quantity using ImageJ and Image Studio software. Analysis of guanylation signal indicated a robust signal in Control and SAM only wells and depressed guanylation signals in GTP and GTP + SAM wells (gel shown is a representative gel). (B) Percent of control was calculated for Control, SAM, GTP and GTP + SAM wells. n = 3.</p

A Sensitive and Robust High-Throughput Screening Assay for Inhibitors of the Chikungunya Virus nsP1 Capping Enzyme

<div><p>Chikungunya virus (CHIKV) is a mosquito-borne <i>Alphavirus</i> that causes severe and debilitating disease symptoms. Alarmingly, transmission rates of CHIKV have increased dramatically over the last decade resulting in 1.7 million suspected cases in the Western hemisphere alone. There are currently no antivirals for treatment of CHIKV infection and novel anti-alphaviral compounds are badly needed. nsP1 is the alphavirus protein responsible for the methyltransferase and guanylyltransferase activities necessary for formation of the 5’ type 0 cap structure added to newly formed viral RNA. Formation of this cap depends on nsP1 binding GTP and transferring a methylated GMP to nascent viral RNA. We have developed a fluorescence polarization-based assay that monitors displacement of a fluorescently-labeled GTP analog in real time. Determining the relative affinities of 15 GTP analogs for nsP1 GTP revealed important structural aspects of GTP that will inform identification of inhibitors able to outcompete GTP for the nsP1 binding site. Validation of the assay for HTS was completed and a secondary orthogonal assay that measures guanylation activity was developed in order to evaluate hits from future drug screens. This platform provides an avenue for identification of potent nsP1 inhibitors, which would potentially provide compounds capable of treating disease caused by CHIKV infection.</p></div

Validation of CHIKV nsP1 purity and identity.

<p>Recombinant CHIKV nsP1 was purified with 6X his-mediated affinity chromatography followed by size exclusion chromatography. (A) Early attempts to purify CHIKV nsP1 resulted in highly aggregated protein that eluted exculsively in the void volume (eluted around 55 mL) during gel filtration, while protein purified after optimization of expression and purification conditions eluted around 72 mL. (B) Eluates resulting from size exclusion chromatography were concentrated and analyzed with SDS-PAGE gel electrophoresis and Coomassie staining. (C) This band was cut out, digested with trypsin and analysed further with orbitrap mass spectrometry analysis.</p

GTP and RNA cap analog competition analysis with CHIKV nsP1.

<p>GTP and RNA cap analog competition analysis with CHIKV nsP1.</p

Motif V is a highly correlated and centralized structure within subdomains 1 and 2.

<p>(A) Vertical segment of the ssRNA+ATP correlation heat map focusing on motif V (residues 407 to 420). Conserved motifs that have strong correlations with motif V are highlighted by horizontal lines on the heat map, colored as shown in the legend. (B) ssRNA+ATP exemplar structure depicting the central position of motif V in relation to the NTPase active site and the conserved motifs highlighted in panel (A). The ATP and lytic water molecules are shown to highlight the proximal location of motif V residues with respect to the NTPase active site.</p

Effects of capping enzyme ligands on RNA binding.

<p><b>A</b>) Effect of purine nucleotides on ppAGUAA-FAM and AGUAA-FAM RNA binding. K_D values for ppAGUAA binding to wild-type dengue capping enzyme were determined in the presence of increasing concentrations of the indicated nucleotide. AGUAA binding was determined only in the presence of 50 µM GTP or Mock. <b>B</b>) Effect of SAM and SAH on ppAGUAA-FAM RNA binding affinity. n = 3.</p

Inhibition of guanylation by GTP analogs.

<p>nsP1 was incubated with GTP-680 and 100 μM of each GTP analog for 1 hour. Reactions were resolved on SDS-PAGE gels, scanned for fluorescence on an Odyssey Clx infared imager, and then stained with Coomassie. Guanylation was quantified and normalized to protein amount using ImageJ and Image Studio software. Percent of control (containing no GTP analog) was calculated for each inhibitor. n = 3.</p

The NTPase cycle of NS3h.

<p>A schematic depicting the hypothesized substrate cycle that NS3h moves through during the NTPase function. Free energy released from this cycle powers the unwinding of dsRNA and unidirectional translocation along the nucleic polymer. The protein structure (inset) demonstrates the tertiary structure of NS3h as well as the positions of the RNA-binding cleft (ssRNA substrate colored blue) and the NTPase active site (ATP molecule colored purple).</p

Energy landscape and structures of the NTP hydrolysis reaction in the active site of DENV NS3h for the ATP (A) and ssRNA+ATP (B) substrate states.

<p>DFT calculations were performed using the <i>ω</i>B97X-D/6-31+G* level of theory. A total of 138 atoms were included in the quantum mechanical calculations (see supporting information for full structures) but only the triphosphate, lytic water, Mg²⁺, and Glu285 side chain atoms are shown here for clarity. Important distances and angles are included in the structural representations of each state. All energies are reported in units of kcal mol⁻¹.</p