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.
Highlights
Human metapneumovirus (HMPV), first isolated in 2001 [1], is a leading cause of lower respiratory infection in infants and children worldwide [2,3,4,5,6,7,8,9,10,11,12,13]
Most paramyxoviruses enter cells by coupling receptor binding to membrane fusion at the cell surface, the entry mechanism for HMPV is largely uncharacterized
We show that HMPV particles are internalized by clathrin-mediated endocytosis and fuse with endosomal membranes
Summary
Human metapneumovirus (HMPV), first isolated in 2001 [1], is a leading cause of lower respiratory infection in infants and children worldwide [2,3,4,5,6,7,8,9,10,11,12,13]. Paramyxovirus membrane fusion is thought to occur at the plasma membrane, largely based on observations that paramyxoviruses fuse in a pH-independent manner and often induce cellcell fusion or syncytia formation in cell culture. For influenza virus and vesicular stomatitis virus (VSV), a drop in pH triggers conformational changes in the viral fusion proteins [15,16]; endosomal entry and acidification are required for productive infection. Paramyxoviruses and most retroviruses are resistant to ammonium chloride, a weak base that blocks vacuolar acidification, suggesting that these viruses induce membrane fusion at neutral pH and do not require endocytosis. PH-independent virus fusion can occur either at the cell surface or after internalization into endosomes, and resistance to acidification inhibitors does not necessarily indicate where virus-cell membrane fusion occurs
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