Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae
of RNA viruses. RSV, human metapneumovirus, pneumonia virus of mice (PVM), and avian pneumoviruses form the subfamily Pneumovirinae
. RSV is a leading cause of pneumonia and bronchiolitis in infants and the elderly and is estimated to cause 30 million lower respiratory tract infections and more than 60,000 deaths worldwide each year (32
). This global health burden could be ameliorated by an effective vaccine, but one does not currently exist. A series of RSV vaccine trials in the 1960s that evaluated an alum-precipitated formalin-inactivated whole virus did not prevent infection and caused enhanced disease severity upon natural RSV infection (11
), in part due to poor elicitation of fusion-inhibiting and neutralizing antibodies (17
). Severe disease caused by RSV can be reduced by monthly injections of the monoclonal antibody palivizumab (Synagis) (21
), but the high cost of treatment restricts its use. Thus, the creation of a safe and effective RSV vaccine is still urgently needed.
RSV-neutralizing antibodies target either the attachment (G) glycoprotein or the fusion (F) glycoprotein (40
). The F glycoprotein is a type I viral fusion protein, a class of entry machines that includes influenza virus hemagglutinin and HIV-1 envelope. Much of our knowledge regarding the structural rearrangements of type I fusion proteins derives from crystal structures of the influenza virus hemagglutinin (7
) and paramyxovirus F (10
) glycoproteins, which have been determined in the pre- and postfusion states. Type I fusion proteins are synthesized as inactive, single-chain polypeptides that assemble into trimers. The proteins become active after cleavage by host proteases, which liberate a hydrophobic stretch of amino acids called the fusion peptide. After binding to the target cell and subsequent activation, the metastable prefusion protein undergoes a series of dramatic structural rearrangements that result in the insertion of the fusion peptide into the target cell membrane, followed by the formation of a stable helical bundle that forms as the viral and cell membranes are apposed (reviewed in reference 12
Unlike most type I viral fusion proteins, the RSV F precursor (F0
) is cleaved by a furin-like protease at two sites, which generates three fragments. The shorter, N-terminal fragment (F2
) is covalently attached to the larger, C-terminal fragment (F1
) by two disulfide bonds. F2
is predicted to have two N-glycosylation sites at Asn27 and Asn70, and F1
is predicted to have one N-glycosylation site at Asn500. The intervening fragment of 27 amino acids is predicted to have 2 or 3 N-glycosylation sites depending on the RSV subtype, but this fragment dissociates after cleavage and is not found in the mature protein (6
). Neutralizing antibodies, such as palivizumab and 101F, target epitopes that have been mapped to linear regions in the F1
subunit, referred to as antigenic site II and site IV, respectively (3
). The structures of the epitope peptides bound to 101F and motavizumab, a potent derivative of palivizumab, have been determined (28
), and both structures suggest that the epitopes are larger than the linear peptides. Modeling of these epitopes on the RSV F glycoprotein has been performed using structures of soluble F ectodomains from paramyxoviruses in the subfamily Paramyxovirinae
), but low sequence homology decreased modeling accuracy and limited conclusions that could be drawn.
To obtain structural information on the RSV F glycoprotein ectodomain, we created a soluble, furin-cleaved ectodomain construct and determined its structure. Here, we present the 2.8-Å crystal structure of the RSV F glycoprotein in the postfusion state. The structure reveals that the 101F and motavizumab epitopes exist in the F glycoprotein in conformations that are similar to the antibody-bound peptide structures. Binding experiments demonstrate that the postfusion state can bind 101F and palivizumab with nanomolar affinity and can bind motavizumab with picomolar affinity. Modeling predicts the full extent of the epitopes and reveals that 101F interactions are contained within a single protomer, whereas motavizumab recognizes residues on two protomers in the trimer. These results are discussed in the context of antibody-mediated RSV neutralization and vaccine design.