JVI Accepts, published online ahead of print on 26 November 2008

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J. Virol. doi:10.1128/JVI.01381-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

LOW PH TRIGGERING OF HUMAN METAPNEUMOVIRUS FUSION: ESSENTIAL RESIDUES AND IMPORTANCE IN ENTRY

Rachel M. Schowalter, Andres Chang, Jessica G. Robach, Ursula J. Buchholz, and Rebecca Ellis Dutch^*

Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536-0509; Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA; Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892-8007, USA

^* To whom correspondence should be addressed. Email: rdutc2{at}uky.edu.

Human metapneumovirus (HMPV) is a significant respiratory pathogen classified in the Pneumovirinae subfamily of the paramyxovirus family. Recently, we demonstrated that HMPV F protein-promoted cell-cell fusion is stimulated by exposure to low pH, in contrast to what is observed for other paramyxovirus F proteins. In the present study, we examined the potential role of histidine protonation in HMPV F fusion, and investigated the role of low pH in HMPV viral entry. Mutagenesis of the three ectodomain histidine residues of the HMPV F protein demonstrated that mutation of a histidine in the heptad repeat B linker domain (H435) ablated fusion activity without altering cell surface expression or proteolytic processing significantly. Modeling of the HMPV F protein revealed several basic residues surrounding this histidine residue, and mutation of these residues also reduced fusion activity. These results suggest that electrostatic repulsion in the HRB linker region may contribute to triggering of HMPV F. In addition, we examined the effect of inhibitors of endosomal acidification or endocytosis on entry of a recombinant GFP-expressing HMPV. Interestingly, chemicals that raise the pH of endocytic vesicles resulted in a 30-50% decrease in HMPV infection, while inhibitors of endocytosis reduced infection by as much as 90%. These data suggest that HMPV utilizes an endocytic entry mechanism, in contrast to what has been hypothesized for most paramyxoviruses. In addition, our results indicate that HMPV uses the low pH of the endocytic pathway to enhance infectivity, though the role of low pH likely differs from classically described mechanisms.

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