Investigating on the binding characteristics of the important RNA‐protein interactions in Brome Mosaic Virus

Abstract

RNA‐protein interactions are often crucial for viability of many biological systems, including the replication cycle of RNA viruses. Understanding molecular driving forces behind the binding specificity of these interactions in RNA viruses is important in advancing our knowledge not only in the mechanism of viral replication but also in designing therapies against viral infections. Here, we have investigated the binding characteristics of the N‐terminal region of a viral Coat Protein (CP) to promoter sequences of the genomic RNA in Brome Mosaic Virus (BMV), a granary plant‐infecting RNA virus. BMV has three separate RNA molecules as its genomic RNA and coordinating the timely production of these three RNAs is crucial in successful replication of their progeny viruses. Previous studies showed that the coordinating function is conducted by the CP, which involves its specific binding on BMV RNA. The N‐terminal region of CP binds to a couple of promoter sequence of BMV genomic RNA. Interestingly those RNA motifs all have well‐defined loop structures. We have explored on the importance of the three‐dimensional conformations of these RNA motifs for the recognition by the N‐terminal region of CP. We designed peptides mimicking the N‐terminal region of CP, called CPNT, and also prepared RNA sequences representing the promoter motifs of BMV RNA. We have used Fluorescence quenching & anisotropy as well as NMR and FPLC for investigating the binding characteristics of CPNT to these RNA. Our initial results have shown that one of the RNA promoter motifs, found at the 5′end of BMV genomic RNA, showed specific binding affinity in which the conformation of its triloop (5′AUA3′) serves a key recognition motif for the CPNT‐RNA interaction. We are currently investigating the binding characteristic of other RNA motifs with CPNT as well as the effects of pH, salt, and temperature on them. Fluorescence spectroscopy and Fast Protein Liquid Chromatography are used to measure and compare binding efficiencies of peptide‐RNA complexes for pH 7.5, 7.0, 6.5, 6.0, and 5.5 in sodium chloride and ammonium acetate, with native polyacrylamide gel electrophoresis (PAGE) as a way to visualize binding of those complexes. High‐resolution 1D and 2D NMR is used to gain structural insights into complexes. These new results will be presented at the conference.Support or Funding InformationThis project was supported by the Center for Student Research and by the Faculty Support Grant from CSU East Bay. A Varian 500 MHz FT‐NMR instrument was obtained with funds from NSF MRI grant 0420615 and the CSU East Bay College of Science.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.