Human Metapneumovirus Is Capable of Entering Cells by Fusion with Endosomal Membranes

• Type of Material: Article
• Authors: Reagan G. Cox, Vanderbilt University; Bernardo Mainou, Emory University; John V. Williams, University of Pittsburgh; Monika Johnson, University of Pittsburgh; Andrew K. Hastings, Vanderbilt University; Jennifer E. Schuster, Children’s Mercy Hospital; Terence S. Dermody, Vanderbilt University
• Language: English
• Date: 2015-12-01
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: © 2015 Cox et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.ppat.1005303
• Title of Journal or Parent Work: PLoS Pathogens
• ISSN: 1553-7366
• Volume: 11
• Issue: 12
• Start Page: e1005303
• End Page: e1005303
• Grant/Funding Information: This work was supported by R01 AI08562 (JVW), R21 AI073697 (JVW), T32 AI7611 (RGC), F32 AI801082 (BAM), and R01 AI32539 (TSD).
• Keywords: MEDIATED ENDOCYTOSIS; Virology; Microbiology; Parasitology; Science & Technology; LOW-PH; VESICULAR STOMATITIS-VIRUS; F-PROTEIN; SYNCYTIAL VIRUS; INFLUENZA-VIRUS; Life Sciences & Biomedicine; RESPIRATORY-TRACT DISEASE; BETA-1-INTEGRIN CYTOPLASMIC TAIL; YOUNG-CHILDREN; GENETIC DIVERSITY
• Research Categories: Biology, Microbiology; Health Sciences, Pathology; Biology, Virology

Abstract

Human metapneumovirus (HMPV), a member of the Paramyxoviridae family, is a leading cause of lower respiratory illness. Although receptor binding is thought to initiate fusion at the plasma membrane for paramyxoviruses, the entry mechanism for HMPV is largely uncharacterized. Here we sought to determine whether HMPV initiates fusion at the plasma membrane or following internalization. To study the HMPV entry process in human bronchial epithelial (BEAS-2B) cells, we used fluorescence microscopy, an R18-dequenching fusion assay, and developed a quantitative, fluorescence microscopy assay to follow virus binding, internalization, membrane fusion, and visualize the cellular site of HMPV fusion. We found that HMPV particles are internalized into human bronchial epithelial cells before fusing with endosomes. Using chemical inhibitors and RNA interference, we determined that HMPV particles are internalized via clathrin-mediated endocytosis in a dynamin-dependent manner. HMPV fusion and productive infection are promoted by RGD-binding integrin engagement, internalization, actin polymerization, and dynamin. Further, HMPV fusion is pH-independent, although infection with rare strains is modestly inhibited by RNA interference or chemical inhibition of endosomal acidification. Thus, HMPV can enter via endocytosis, but the viral fusion machinery is not triggered by low pH. Together, our results indicate that HMPV is capable of entering host cells by multiple pathways, including membrane fusion from endosomal compartments.