Flaviviruses are insect-transmitted human pathogens that most notably cause Zika-associated microcephaly, dengue fever and West Nile fever. This study aims to elucidate the molecular mechanisms of the flaviviral nonstructural protein 1 (NS1), a multi-functional virulence protein. Intracellular NS1 is essential to replication of the viral RNA genome, whereas secreted NS1 interacts with innate immunity proteins and, in some cases, induces disease phenotypes. Our crystal structures of full-length, glycosylated NS1 from Zika virus (ZIKV), dengue virus serotype 2 (DENV2) and West Nile virus (WNV) will guide experiments to identify which of the distinct domains of NS1 are responsible for which of its several functions. Despite their overall similarity (~50% identical amino acid sequences), several properties specific to individual flaviviruses are attributed to the NS1 proteins, making comparative analysis especially powerful in dissecting the molecular functions of NS1. An extensive panel of mutants based on comparative mutagenesis of DENV2 and WNV NS1 will be expanded to include ZIKV NS1. Based on observations in the initial comparative study that NS1 affects virus particle assembly, we will test the hypothesis that NS1 acts as an infectivity factor by aiding viral structural protein folding or virus particle transit through the secretory system. Intracellular NS1 is localized to the ER lumen as a membrane-associated dimer with a critical role in replication through association with viral transmembrane proteins. We will probe the interaction with viral protein NS4B through mutagenesis and biophysical experiments. Electron cryo-microscopy or crystallography will be used to investigate the structure of secreted NS1, a hexameric lipo-protein particle. An initial observation that NS1 remodels membranes will be followed by detailed experiments using light microscopy with fluorescently tagged lipids to determine any lipid preference in this key association. Binding experiments will identify which domains of NS1 interact with which domains of two proteins of the complement system and the innate immunity Toll-like receptor 4. The results will provide a foundation for development of antiviral drugs and/or effective vaccines, which are not available or of limited use for Zika, dengue or West Nile viruses.

Public Health Relevance

Flaviviruses are human pathogens that cause Zika-associated microcephaly, dengue fever and West Nile fever. This study will examine how the viral protein known as NS1 aids virus propagation, disrupts normal cellular functions, and helps the virus evade the immune system. The 3D structure of NS1 will guide biological experiments to identify which parts of NS1 are responsible for which of its several functions, providing a foundation for development of antiviral drugs and/or effective vaccines, which are not available for Zika, dengue or West Nile viruses.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
High Priority, Short Term Project Award (R56)
Project #
1R56AI130130-01
Application #
9542638
Study Section
Virology - B Study Section (VIRB)
Program Officer
Challberg, Mark D
Project Start
2017-08-16
Project End
2018-07-31
Budget Start
2017-08-16
Budget End
2018-07-31
Support Year
1
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109