Preparation and Structural Analysis of the Human Respiratory Syncytial Virus M2-2 Protein Público

Abstract

The human respiratory syncytial virus (hRSV) is a non-segmented negative strand (NNS) RNA virus that leads to respiratory infection, particularly in infants and the elderly. Despite its heavy public health burden, an efficient vaccine has not yet been developed. The viral genome is encapsidated by the nucleoprotein N, RNA polymerase L, and cofactors P and M2-1 to form the helical ribonucleocapsid. The M2-2 protein, encoded by the second open reading frame (ORF) of the M2 gene, is responsible for ribonucleocapsid rearrangement and for favoring viral replication over transcription. While many of the structural studies on RSV proteins have been on the G and F membrane proteins and the ribonucleocapsid proteins, there is no structural data on M2-2.

To better understand the function of M2-2 and the mechanism behind its activity, we demonstrate the expression and purification of M2-2 and maltose-binding protein (MBP)- tagged M2-2. We report MBP-M2-2 expression in E. coli competent cells, and using affinity chromatography, ion exchange, and size exclusion chromatography, we were able to obtain pure samples of both proteins. We also describe sitting-drop and hanging-drop vapor diffusion crystallization experiments which the show growth of microcrystals. Finally, we used dynamic light scattering and negative stain electron microscopy to characterize purified M2-2 and MBP- M2-2. This data takes the first step toward solving the structure of M2-2. Structural data on M2-2 will reveal its function at a biochemical level, deepening our understanding of viral RNA synthesis and providing an avenue for vaccine development. 

Table of Contents

Introduction.............................................................................................................................1

Results.....................................................................................................................................4

Expression and Purification of MBP-M2-2...........................................................................4

Purification of Untagged M2-2........................................................................................... 8

MBP-M2-2 Crystal Screen and Optimization...................................................................... 11

DLS and Negative Stain EM of M2-2 and MBP-M2-2 .......................................................... 13

Methods and Materials .............................................................................................................16

E. coli Transformation and MBP-M2-2 Expression ............................................................. 16

MBP-M2-2 Purification.................................................................................................... 17

M2-2 Purification ............................................................................................................ 18

Crystal Screening and Optimization ................................................................................. 19

Dynamic Light Scattering................................................................................................. 20

Negative Stain Electron Microscopy................................................................................... 20

Discussion and Future Directions...............................................................................................21

Supplementary Figures..............................................................................................................24

References ................................................................................................................................28 

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Molecular Chemistry, Biochemistry Biology, Virology
Palavra-chave	Cryo-EM Respiratory Syncytial Virus (RSV) Viral RNA Synthesis X-ray Crystallography
Committee Chair / Thesis Advisor	Liang, Bo, Emory University
Committee Members	Kelly, William, Emory University McKnelly, Kate, Emory University

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