Structural interactions of human respiratory syncytial virus NS2 with human innate immune system receptors RIG-I and MDA5 Restricted; Files Only

Abstract

Respiratory syncytial virus (RSV) is a significant public health threat that causes severe bronchitis and pneumonia in children and immunocompromised individuals. In response to RSV infection, host cells employ various pattern recognition receptors (PRR). These include receptors of the retinoic acid-inducible gene I-like receptors family (RLR) of the innate immune system. retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are two such RLRs that detect cytosolic viral RNA. RSV has 10 genes that code for 11 proteins, including nonstructural protein 2 (NS2) which plays an important role in antagonizing human type I interferon (IFN) response through direct interaction with RIG-I at its caspase recruitment domains (CARD). NS2 is also found to interact with MDA5 in its inactive conformation. Despite these insights into the molecular mechanism of NS2 antagonism, definite structures of the direct interactions of NS2 with the two RLR are not established. In order to address this knowledge gap, we aim to solve the structure of the direct interactions of NS2 with RIG-I and MDA5. First, we optimized an in vitro expression system using E. coli to express recombinant NS2, RIG-I, and MDA5 with a His-MBP tag. We also expressed the CARDs of RIG-I and MDA5 along with our tags in order to examine possible interaction at the domains. The recombinant proteins were purified using Ni-NTA affinity column, ion exchange column, and size exclusion columns. Purified NS2 and MDA5 CARDs were used in pre-crystallization tests to determine suitable conditions of crystallization of the complex between NS2 and MDA5 CARDs.

Table of Contents

Table of Contents

Introduction................................................................................................................................1 Results.......................................................................................................................................... 5

MDA5 full-length purification.............................................................................................5

MDA5 CARDs purification... ...........................................................................................10

NS2-MBP purification.......................................................................................................13

Crystal Conditions Screening ...........................................................................................17

Discussion ............................................................................................................................... 18

Material and method....................................... .....................................................................20

Cells and Plasmids......... ...................................................................................................20

Transformation...................................................................................................................21

Protein expression..............................................................................................................21

Protein purification............ ..............................................................................................21

References...... .......................................................................................................................... 23

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Biology, Molecular Chemistry, Biochemistry
Mot-clé	protein purification MDA5 RIG-I Structural interaction NS2 RSV
Committee Chair / Thesis Advisor	Liang, Bo, Emory University
Committee Members	Gardner, Matthew, Emory University Thakur, Jitendra , Emory University

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