Analysis of RNA Binding by the Dengue Virus NS5 RNA Capping Enzyme

Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5′ end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the KD for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5′ phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5′ di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented

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

RNA binding residues on the dengue capping enzyme.

<p>All residues that were tested in this study were mapped on the dengue virus capping enzyme structure (2P1D) bound to GTP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025795#pone.0025795-Geiss1" target="_blank">[11]</a>. <b>A</b>) Residues that showed greater than 5-fold reduction in RNA binding affinity against AGUAA, pAGUAA, or ppAGUAA are colored in green. Residues that showed less than 5-fold reduction in binding affinity against AGUAA, pAGUAA, or ppAGUAA are colored in magenta. Bound GTP and SAH are shown. <b>B</b>) Surface representation of 2P1D with RNA binding residues colored green and non-binding residues colored magenta.</p

Comparison of dengue, yellow fever, and West Nile virus capping enzyme K_D values for ppAGUAA-FAM RNA.

<p>50 nM ppAGUAA-FAM RNA was incubated in increasing concentrations of wild-type capping enzyme for 1 hr, then fluorescence polarization signal was detected. Curve fits and K_D values were determined with the KaleidaGraph software package as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025795#s2" target="_blank">methods</a> section. n = 3.</p

Comparison of RNA and GTP binding affinities.

<p>Fold change was determined for RNA binding by comparing each mutant K_D value to wild-type (WT) ligand binding value from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025795#pone-0025795-t001" target="_blank">Table 1</a>.</p

Structural superposition of the Vaccinia virus VP39 with the dengue virus capping enzyme.

<p><b>A</b>) Global overlap of 1AV6 (VP39) and 2P1D (dengue virus capping enzyme). Superposition was performed using the TopMatch webserver, and figures were generated in PyMol. Red/orange indicate structural overlap between 1AV6 and 2P1D. Non-overlapping regions are not shown. Bound GTP/Cap and SAH are shown. RNA has been removed for clarity. <b>B</b>) Overlay of bound ligands from 1AV6 (green) and 2P1D (magenta). <b>C</b>) Overlap of 1AV6 residue K32 (interacting with RNA phosphate #6) to 2P1D residues K29, K30, and R212. <b>D</b>) Overlap of 1AV6 residues K41 (interacting with RNA phosphate #4) and K175 (interacting with RNA ribose #1 hydroxyl) to 2P1D K62 and K181, respectively.</p

Effects of 5′ RNA phosphates on dengue capping enzyme binding affinity.

<p>50 nM of AGUAA-FAM, pAGUAA-FAM, and ppAGUAA-FAM was incubated with increasing concentrations of wild-type dengue capping enzyme for 1 hr, then fluorescence polarization signal was detected. n = 3.</p

Lactococcus Lactis Subsp. cremoris Is an Efficacious Beneficial Bacterium that Limits Tissue Injury in the Intestine

Summary: The use of beneficial bacteria to promote health is widely practiced. However, experimental evidence corroborating the efficacy of bacteria promoted with such claims remains limited. We address this gap by identifying a beneficial bacterium that protects against tissue damage and injury-induced inflammation in the gut. We first employed the Drosophila animal model to screen for the capacity of candidate beneficial bacteria to protect the fly gut against injury. From this screen, we identified Lactococcus lactis subsp. cremoris as a bacterium that elicited potent cytoprotective activity. Then, in a murine model, we demonstrated that the same strain confers powerful cytoprotective influences against radiological damage, as well as anti-inflammatory activity in a gut colitis model. In summary, we demonstrate the positive salutary effects of a beneficial bacterium, namely, L. lactis subsp. cremoris on intestinal tissue and propose the use of this strain as a therapeutic to promote intestinal health. : Biological Sciences; Microbiology; Cell Biology Subject Areas: Biological Sciences, Microbiology, Cell Biolog