A biochemical study of the formation of the M2-1 protein tetramer of human respiratory syncytial virus

Abstract

Human Respiratory Syncytial Virus (hRSV) is the major causative agent of acute respiratory infections in children and imunucompromised patients. Belonging to the Paramyxoviridae family RSV is an enveloped virus with helicoidalnucleocapsid. Its genome is constituted of a RNA single-stranded negative polarity, non-segment compose of approximately 15,200 nucleotides and 10 genes, which encode 11 proteins. One of the factors that contribute to the success of the viral replication is the M2-1 protein that acts by interaction with RNA and participates on the replication process, besides being active only in tetrameric form. In this way, to understand the process of tetramerization of the protein M2-1 and to look for conditions to prevent its interaction with the RNA, and an important step to prevent the success of the viral replication. The objectives of this work are to investigate the process of tetramerization of M2-1 protein under different physicochemical conditions by fluorescence spectroscopy and fluorescence anisotropy. To achieve these objectives, will be performed expression and purification of hRSV M2-1 protein and verification of its tetramerization form under different conditions of pH, salt concentration and temperature and Interference with RNA binding. The gene cloned into vector pET28A will be expressed in Escherichia coli bacterium of line BL21. The expressed protein will be purified on affinity chromatography. The tetramerization process will be evaluated through the circular dichroism technique and fluorescence anisotropy varying the saline concentration, temperature and pH, it will be verified the interference of these conditions for the affinity of the RNA to the M2-1 protein, the monomer affinity constants for tetramer formation and RNA binding under different physicochemical conditions will be calculated. The results of the present study will allow to identify the mechanism of tetramerization of the M2-1 protein, allowing to obtain an interaction model as well as propose therapeutic models. (AU)