Transient Changes of the Conformation of Hemagglutinin of Influenza Virus at Low pH Detected by Time-resolved Circular Dichroism Spectroscopy

Thomas Korte,Kai Ludwig,Mathias Krumbiegel,Dietrich Zirwer,Gregor Damaschun,Andreas Herrmann

doi:10.1074/jbc.272.15.9764

Abstract

Membrane fusion of influenza virus is mediated by a conformational change of the viral membrane protein hemagglutinin (HA) triggered by low pH. By near UV CD spectroscopy, which is sensitive to the arrangement and mobility of aromatic amino acids in proteins, we have monitored continuously with a time resolution of 5 s the kinetics of structural alterations of the ectodomain of HA isolated from different influenza virus strains (H1 (A/PR 8/34), H2 (A/Japan), and H3 (X31)). To establish a functional correlation to structural alterations of the HA ectodomain reflected by the CD, we have measured the kinetics of the virus-erythrocyte fusion and of the inactivation of fusion by low pH preincubation of viruses. At acidic pH we found a multiphasic behavior of the CD signal recorded at 283 nm. Upon lowering the pH we detected first an increase of the CD amplitude, which is associated with the formation of a fusion-competent state of HA. The initial increase was followed by a continuous decline of CD amplitude, which can be ascribed to a transformation into a fusion-inactivated conformation that is in its early phase reversible as found for A/Japan. The half-time of the different phases of the CD signal depended on the virus strain, the temperature, and the acidic pH. The results support recent hypotheses that the fusion-competent conformation is an intermediate of the fusion-inactivated structure of HA.

Highlights

The fusion of the viral and the endosomal membranes subsequent to the endocytic uptake of influenza virus is mediated by the integral membrane protein hemagglutinin (HA).1 HA is a homotrimeric glycoprotein that consists of the two subunits, HA1, bearing the receptor binding site, and HA2, anchored with its C terminus in the viral membrane
In the present study we have investigated whether acidinduced conformational changes of hemagglutinin leading either to the activation or inactivation of the fusogenic properties of HA, respectively, can be distinguished by CD spectroscopy in far and near UV
Near UV CD spectra of HA from various strains measured at pH 7.4, 37 °C. At pH 5.4 (37 °C), were comparable to those reported previously for X31 by Wharton et al [28]

Summary

Introduction

The fusion of the viral and the endosomal membranes subsequent to the endocytic uptake of influenza virus is mediated by the integral membrane protein hemagglutinin (HA). HA is a homotrimeric glycoprotein that consists of the two subunits, HA1, bearing the receptor binding site, and HA2, anchored with its C terminus in the viral membrane. In the present study we have investigated whether acidinduced conformational changes of hemagglutinin leading either to the activation or inactivation of the fusogenic properties of HA, respectively, can be distinguished by CD spectroscopy in far and near UV To this end HA trimers were purified from various influenza strains belonging to different subtypes (H1 (A/PR 8/34); H2 (A/Japan/305/57); H3 (X31)) by detergent extraction. Comparing these results to the time dependence of viral fusion with erythrocyte membranes and of the low pHmediated inactivation of fusion activity, we were able to relate distinct phases of the structural rearrangements detected by near UV CD to the formation of a fusion-competent state and of

Methods

Results

Conclusion