Five Residues in the Apical Loop of the Respiratory Syncytial Virus Fusion Protein F ₂ Subunit Are Critical for Its Fusion Activity

Abstract

The respiratory syncytial virus (RSV) fusion (F) protein is a trimeric, membrane-anchored glycoprotein capable of mediating both virus-target cell membrane fusion to initiate infection and cell-cell fusion, even in the absence of the attachment glycoprotein. The F protein is initially expressed in a precursor form, whose functional capabilities are activated by proteolysis at two sites between the F and F subunits. This cleavage results in expression of the metastable and high-energy prefusion conformation. To mediate fusion, the F protein is triggered by an unknown stimulus, causing the F subunit to refold dramatically while F changes minimally. Hypothesizing that the most likely site for interaction with a target cell component would be the top, or apex, of the protein, we determined the importance of the residues in the apical loop of F by alanine scanning mutagenesis analysis. Five residues were not important, two were of intermediate importance, and all four lysines and one isoleucine were essential. Alanine replacement did not result in the loss of the pre-F conformation for any of these mutants. Each of the four lysines required its specific charge for fusion function. Alanine replacement of the three essential lysines on the ascent to the apex hindered fusion following a forced fusion event, suggesting that these residues are involved in refolding. Alanine mutations at Ile64, also on the ascent to the apex, and Lys75 did not prevent fusion following forced triggering, suggesting that these residues are not involved in refolding and may instead be involved in the natural triggering of the F protein. RSV infects virtually every child by the age of 3 years, causing nearly 33 million acute lower respiratory tract infections (ALRI) worldwide each year in children younger than 5 years of age (H. Nair et al., Lancet 375:1545-1555, 2010). RSV is also the second leading cause of respiratory system-related death in the elderly (A. R. Falsey and E. E. Walsh, Drugs Aging 22:577-587, 2005; A. R. Falsey, P. A. Hennessey, M. A. Formica, C. Cox, and E. E. Walsh, N Engl J Med 352:1749-1759, 2005). The monoclonal antibody palivizumab is approved for prophylactic use in some at-risk infants, but healthy infants remain unprotected. Furthermore, its expense limits its use primarily to developed countries. No vaccine or effective small-molecule drug is approved for preventing disease or treating infection (H. M. Costello, W. Ray, S. Chaiwatpongsakorn, and M. E. Peeples, Infect Disord Drug Targets, 12:110-128, 2012). The essential residues identified in the apical domain of F are adjacent to the apical portion of F, which, upon triggering, refolds into a long heptad repeat A (HRA) structure with the fusion peptide at its N terminus. These essential residues in F are likely involved in triggering and/or refolding of the F protein and, as such, may be ideal targets for antiviral drug development.

Small molecule inhibits respiratory syncytial virus entry and infection by blocking the interaction of the viral fusion protein with the cell membrane

“…In the first series of experiments, we investigated whether 3,4-DCQAME affects the function of F protein, which facilitates viral entry by inducing fusion to the cellular membrane. As reported previously, soluble F protein can maintain in a pretriggered state in a high molarity buffer (21,29,30). In our experiments, recombinant RSV F(ecto) protein (Sino Biological), which encodes the ectodomain of F protein, was expressed and purified in a high ionic-strength buffer (20 mM Tris, 500 mM NaCl, PH 7.4, 10% Glycerinum), and then incubated in the absence or presence of 3,4-DCQAME.…”

Transient opening of trimeric prefusion RSV F proteins

“…The C-terminal F 1 subunit contains the transmembrane domain, two heptad repeats, and an N-terminal fusion peptide. Residues in the F 2 subunit contribute to fusogenicity of the F protein and possibly the species specificity of RSV 31–35 . In the mature protein, the F 1 and F 2 subunits are covalently associated via two disulfide bonds 10,11,30 .…”

Mathematical Modeling Identifies the Role of Adaptive Immunity as a Key Controller of Respiratory Syncytial Virus (RSV) Titer in Cotton Rats

“…We employed a non-linear mixed effects approach that models age-specific parameters as random effects. For each time point t in (1,2,3,4,5,6,7,9,12,15, and, 20) days post inoculation and for each age a, we computed the logarithm of the geometric mean RSV titer (denoted log 10 V (data) (t,a)) at a particular time t, where the geometric mean was taken over 3 cotton rats in an animal cohort of age a. The parameters of the non-linear mixed effects model were estimated by maximum likelihood (ML), which (provided that there is a unique maximum) finds the set of parameter values that makes the RSV titer log 10 .…”