Abstract
Human metapneumovirus (hMPV) is a frequent cause of bronchiolitis in young children. Its F glycoprotein mediates virus–cell membrane fusion and is the primary target of neutralizing antibodies. The inability to produce recombinant hMPV F glycoprotein in the metastable pre-fusion conformation has hindered structural and immunological studies. Here, we engineer a pre-fusion-stabilized hMPV F ectodomain and determine its crystal structure to 2.6 Å resolution. This structure reveals molecular determinants of strain-dependent acid-induced fusion, as well as insights into refolding from pre- to post-fusion conformations. A dense glycan shield at the apex of pre-fusion hMPV F suggests that antibodies against this site may not be elicited by host immune responses, which is confirmed by depletion studies of human immunoglobulins and by mouse immunizations. This is a major difference with pre-fusion F from human respiratory syncytial virus (hRSV), and collectively our results should facilitate development of effective hMPV vaccine candidates.
Highlights
Human metapneumovirus is a frequent cause of bronchiolitis in young children
The bestexpressed variant from transfected CV-1 cells was 115-BV, as measured by reactivity with monoclonal antibody MF14, an antibody that recognizes an epitope equivalent to human respiratory syncytial virus (hRSV) F antigenic site II, which is shared by pre-fusion and post-fusion structures
A single proline substitution in combination with modifications to the fusion-activating trypsin cleavage site and the addition of a foldon trimerization domain at the C-terminus of the Human metapneumovirus (hMPV) F ectodomain generated a soluble construct with antigenic characteristics of the pre-fusion conformation
Summary
Human metapneumovirus (hMPV) is a frequent cause of bronchiolitis in young children. Its F glycoprotein mediates virus–cell membrane fusion and is the primary target of neutralizing antibodies. The pre-fusion-to-postfusion transition includes refolding of heptad repeat A (HRA) sequences of the F1 subunit into one long α-helix, and insertion of the fusion peptide—located at the N-terminus of HRA—into the target-cell membrane. Refolding of this fusion intermediate promotes assembly of HRA and HRB sequences into a stable sixhelix bundle that drives membrane fusion and is characteristic of post-fusion F. The structural studies with hRSV F identified antigenic sites unique to the pre-fusion conformation[28, 31] that are recognized by potent neutralizing antibodies that account for most of the neutralizing activity found in human serum[32, 33]. There are neutralizing antibodies that bind epitopes shared by pre-fusion and post-fusion hRSV F, explaining the capacity of purified post-fusion hRSV F to induce neutralizing antibodies and afford protection against hRSV challenge in mice[30]
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