Quantifying the Specific Binding between West Nile Virus Envelope Domain III Protein and the Cellular Receptor αVβ3 Integrin

Justin Jang-Hann Chu,Mah-Lee Ng,Jason Wei-Ming Lee

doi:10.1074/jbc.m506614200

Justin Jang-Hann Chu, Mah-Lee Ng + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m506614200

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Abstract

A previous study has illustrated that the alphaVbeta3 integrin served as the functional receptor for West Nile virus (WNV) entry into cells. Domain III (DIII) of WNV envelope protein (E) was postulated to mediate virus binding to the cellular receptor. In this study, the specificity and affinity binding of WNV E DIII protein to alphaVbeta3 integrin was confirmed with co-immunoprecipitation and receptor competition assay. Binding of WNV E DIII protein to alphaVbeta3 integrin induced the phosphorylation of focal adhesion kinase that is required to mediate ligand-receptor internalization into cells. A novel platform was then developed using the atomic force microscopy to measure this specific binding force between WNV E DIII protein and the cellular receptor, alphaVbeta3 integrin. The single protein pair-interacting force measured was in the range of 45 +/- 5 piconewtons. This interacting force was highly specific as minimal force was measured in the WNV E DIII protein interaction with alphaVbeta5 integrin molecules and heparan sulfate. These experiments provided an insight to quantitate virus-receptor interaction. Force measurement using atomic force microscopy can serve to quantitatively analyze the effect of candidate drugs that modulate virus-host receptor affinity.

Highlights

Crystallography data on the ectodomain of the Flavivirus E protein reveals three distinct domains: a central domain designated as domain I (DI), an elongated dimerization region designated as domain II (DII), and domain III (DIII), having an immunoglobulin-like constant domain [3]
West Nile virus (WNV) has been shown to bind with ␣V␤3 integrin on the surface of host cells and triggered the integrin-associated outside-in signaling that facilitates the infectious entry of the virus into cells [8]
The domain III of the WNV E protein was identified as the virus attachment domain [7]

Summary

Introduction

Crystallography data on the ectodomain of the Flavivirus E protein reveals three distinct domains: a central domain designated as domain I (DI), an elongated dimerization region designated as domain II (DII), and domain III (DIII), having an immunoglobulin-like constant domain [3] Both DII and DIII of the E protein have been suggested to be important for binding to the cellular receptor (4 –7). In this study the specific interaction between the DIII of WNV E protein and its cellular receptor (␣V␤3 integrin) was first analyzed, and subsequently, atomic force microscope (AFM) was used to directly measure the interaction forces between the viral and cellular proteins. The probe is gradually pulled away (retract phase; Supplemental Fig. 1c), and at this stage there is a repulsive force due to the cantilever bending. This study provides a novel insight to the understanding of the specific interaction between of WNV receptor attachment domain (E DIII protein) and ␣V␤3 integrin. A platform for efficacy testing of putative anti-viral compounds that can disrupt

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