Characterization of human metapneumovirus F protein-promoted membrane fusion: critical roles for proteolytic processing and low pH.
Human metapneumovirus (HMPV) is a recently described human pathogen of the pneumovirus subfamily within the paramyxovirus family. HMPV infection is prevalent worldwide and is associated with severe respiratory disease, particularly in infants. The HMPV fusion protein (F) amino acid sequence contains features characteristic of other paramyxovirus F proteins, including a putative cleavage site and potential N-linked glycosylation sites. Propagation of HMPV in cell culture requires exogenous trypsin, which cleaves the F protein, and HMPV, like several other pneumoviruses, is infectious in the absence of its attachment protein (G). However, little is known about HMPV F-promoted fusion, since the HMPV glycoproteins have yet to be analyzed separately from the virus. Using syncytium and luciferase reporter gene fusion assays, we determined the basic requirements for HMPV F protein-promoted fusion in transiently transfected cells. Our data indicate that proteolytic cleavage of the F protein is a stringent requirement for fusion and that the HMPV G protein does not significantly enhance fusion. Unexpectedly, we also found that fusion can be detected only when transfected cells are treated with trypsin and exposed to low pH, indicating that this viral fusion protein may function in a manner unique among the paramyxoviruses. We also analyzed the F protein cleavage site and three potential N-linked glycosylation sites by mutagenesis. Mutations in the cleavage site designed to facilitate endogenous cleavage did so with low efficiency, and our data suggest that all three N-glycosylation sites are utilized and that each affects cleavage and fusion to various degrees.
Schowalter
Rachel M
RM
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Biomedical Biological Sciences Research Building, 741 South Limestone, Lexington, KY 40536-0509, USA.
Smith
Stacy E
SE
Dutch
Rebecca Ellis
RE
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D017930:Genes, Reporter; D015726:Giant Cells / Q000821:virology; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008561:Membrane Fusion / Q000235:genetics / Q000502:physiology*; D029121:Metapneumovirus / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D011499:Protein Processing, Post-Translational; D014357:Trypsin / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D014760:Viral Fusion Proteins; D008156:Luciferases; D014357:Trypsin