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Journal of Virology, October 2007, p. 10758-10768, Vol. 81, No. 19
0022-538X/07/$08.00+0     doi:10.1128/JVI.00725-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Role of Endocytosis and Low pH in Murine Hepatitis Virus Strain A59 Cell Entry{triangledown}

Patricia Eifart,1 Kai Ludwig,2 Christoph Böttcher,2 Cornelis A. M. de Haan,3 Peter J. M. Rottier,3 Thomas Korte,1* and Andreas Herrmann1*

Humboldt-Universität zu Berlin, Institut für Biologie/Biophysik, Mathematisch-Naturwissenschaftliche Fakultät I, Berlin, Germany,1 Forschungszentrum für Elektronenmikroskopie, Freie Universität Berlin, Berlin, Germany,2 Utrecht University, Faculty of Veterinary Medicine, Department of Infectious Diseases and Immunology, Utrecht, The Netherlands3

Received 4 April 2007/ Accepted 29 June 2007

Infection by the coronavirus mouse hepatitis virus strain A59 (MHV-A59) requires the release of the viral genome by fusion with the respective target membrane of the host cell. Fusion is mediated by the viral S protein. Here, the entry pathway of MHV-A59 into murine fibroblast cells was studied by independent approaches. Infection of cells assessed by plaque reduction assay was strongly inhibited by lysosomotropic compounds and substances that interfere with clathrin-dependent endocytosis, suggesting that MHV-A59 is taken up via endocytosis and delivered to acidic endosomal compartments. Infection was only slightly reduced in the presence of substances inhibiting proteases of endosomal compartments, precluding that the endocytic uptake is required to activate the fusion potential of the S protein by its cleavage. Fluorescence confocal microscopy of labeled MHV-A59 confirmed that virus is taken up via endocytosis. Bright labeling of intracellular compartments suggests their fusion with the viral envelope. No fusion with the plasma membrane was observed at neutral pH conditions. However, when virus was bound to cells and the pH was lowered to 5.0, we observed a strong labeling of the plasma membrane. Electron microscopy revealed low pH triggered conformational alterations of the S ectodomain. Very likely, these alterations are irreversible because low-pH treatment of viruses in the absence of target membranes caused an irreversible loss of the fusion activity. The results imply that endocytosis plays a major role in MHV-A59 infection and the acidic pH of the endosomal compartment triggers a conformational change of the S protein mediating fusion.

* Corresponding authors. Mailing address: Institut für Biologie/Biophysik, Humboldt-Universität zu Berlin, Invalidenstr. 42, D-10115 Berlin, Germany. Fax: 49 30 2093 8585. E-mail for T. Korte: thomas.korte{at}rz.hu-berlin.de. E-mail for A. Herrmann: andreas.herrmann{at}rz.hu-berlin.de

{triangledown} Published ahead of print on 11 July 2007.

Journal of Virology, October 2007, p. 10758-10768, Vol. 81, No. 19
0022-538X/07/$08.00+0     doi:10.1128/JVI.00725-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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