title: Human Metapneumovirus Is Capable of Entering Cells by Fusion with Endosomal Membranes.
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.
authors.affiliation: Department of Pathology, Microbiology, & Immunology, Division of Infectious Diseases, Vanderbilt University School of Medicine; Nashville, Tennessee, United States of America.
authors.forename: Reagan G
authors.initials: RG
authors.lastname: Cox
authors.affiliation: Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors.forename: Bernardo A
authors.initials: BA
authors.lastname: Mainou
authors.affiliation: Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Monika
authors.initials: M
authors.lastname: Johnson
authors.affiliation: Department of Pathology, Microbiology, & Immunology, Division of Infectious Diseases, Vanderbilt University School of Medicine; Nashville, Tennessee, United States of America.
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Hastings
authors.affiliation: Department of Pediatrics, Children's Mercy Hospital, Kansas City, Missouri, United States of America.
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Schuster
authors.affiliation: Department of Pathology, Microbiology, & Immunology, Division of Infectious Diseases, Vanderbilt University School of Medicine; Nashville, Tennessee, United States of America.|Department of Pediatrics, Division of Infectious Diseases, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.
authors.forename: Terence S
authors.initials: TS
authors.lastname: Dermody
authors.affiliation: Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States of America.
authors.forename: John V
authors.initials: JV
authors.lastname: Williams
mesh_terms: D001980:Bronchi / Q000821:virology; D002460:Cell Line; D011992:Endosomes / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D029121:Metapneumovirus / Q000502:physiology*; D018613:Microscopy, Confocal; D018184:Paramyxoviridae Infections / Q000378:metabolism*; D034741:RNA, Small Interfering; D020545:Respiratory Mucosa / Q000821:virology*; D014162:Transfection; D014760:Viral Fusion Proteins / Q000378:metabolism; D053586:Virus Internalization
chemical_list: D034741:RNA, Small Interfering; D014760:Viral Fusion Proteins