The Heptad Repeat C Domain of the Respiratory Syncytial Virus Fusion Protein Plays a Key Role in Membrane Fusion

Abstract

Respiratory syncytial virus (RSV) mediates host cell entry through the fusion (F) protein, which undergoes a conformational change to facilitate the merger of viral and host lipid membrane envelopes. The RSV F protein comprises a trimer of disulfide-bonded F 1 and F 2 subunits that is present on the virion surface in a metastable prefusion state. This prefusion form is readily triggered to undergo refolding to bring two heptad repeats (heptad repeat A [HRA] and HRB) into close proximity to form a six-helix bundle that stabilizes the postfusion form and provides the free energy required for membrane fusion. This process can be triggered independently of other proteins. Here, we have performed a comprehensive analysis of a third heptad repeat region, HRC (amino acids 75 to 97), an amphipathic ␣-helix that lies at the interface of the prefusion F trimer and is a major structural feature of the F 2 subunit. We performed alanine scanning mutagenesis from Lys-75 to Met-97 and assessed all mutations in transient cell culture for expression, proteolytic processing, cell surface localization, protein conformation, and membrane fusion. Functional characterization revealed a striking distribution of activity in which fusion-increasing mutations localized to one side of the helical face, while fusion-decreasing mutations clustered on the opposing face. Here, we propose a model in which HRC plays a stabilizing role within the globular head for the prefusion F trimer and is potentially involved in the early events of triggering, prompting fusion peptide release and transition into the postfusion state.IMPORTANCE RSV is recognized as the most important viral pathogen among pediatric populations worldwide, yet no vaccine or widely available therapeutic treatment is available. The F protein is critical for the viral replication process and is the major target for neutralizing antibodies. Recent years have seen the development of prefusion stabilized F protein-based approaches to vaccine design. A detailed understanding of the specific domains and residues that contribute to protein stability and fusion function is fundamental to such efforts. Here, we present a comprehensive mutagenesis-based study of a region of the RSV F 2 subunit (amino acids 75 to 97), referred to as HRC, and propose a role for this helical region in maintaining the delicate stability of the prefusion form.KEYWORDS respiratory syncytial virus, fusion, membrane fusion, mutagenesis, heptad repeat R espiratory syncytial virus (RSV) is widely considered the most significant viral pediatric pathogen worldwide. Belonging to the Pneumovirinae family, RSV causes substantial morbidity and mortality worldwide, with an estimated 33.8 million acute lower respiratory tract infections per year in children under 5 years of age, and has been linked to almost 200,000 deaths annually, 99% of which are in developing countries (1, 2). RSV is also recognized as an important pathogen of high-risk adult and elderly populations (3). Despite this, no targeted drug or vaccin...

Genotyping of Type A Human Respiratory Syncytial Virus Based on Direct F Gene Sequencing

“…F2 possesses heptad repeat C (HRC), while F1 possesses, at its N terminal, a hydrophobic fusion peptide (FP) followed by two heptad repeats, A and B (HRA and HRB). HRA, HRB, and HRC are essential for envelope fusion to the host cell membrane [12,13]. F2 possesses five N-glycosylation sites, while HRB, part of F1 , possesses a single N-glycosylation site; however, F2 contains more conserved sequence than F1, which is characterized by a high homology among different hRSV genotypes [14].…”

Intranasal vaccination with a recombinant protein CTA1-DD-RBF protects mice against hRSV infection

“…The highly conserved F protein can induce antibodies against infections caused by hRSV of both subgroups A and B 9 and is therefore a key target in the development of subunit vaccines, particle-like vaccines and viral vector-based vaccines 10 – 12 . The F protein is initially expressed as an uncleaved F0 precursor that is activated by furin cleavage at two sites into the mature prefusion F protein (pre-F) 13 , 14 . The F protein is a trimeric glycoprotein that transitions between a pre-F conformation and a postfusion structure to facilitate hRSV entry into target cells 15 , 16 .…”

Identification of Respiratory Syncytial Virus Fusion Protein Inhibitor: In silico Screening and Molecular Docking Approach

“…Human RSV disease pathogenesis is still not well described and the pathways associated with many diseases caused by RSV infection have not been delineated [9]. Initially, the F protein is expressed as a precursor (F0) that is broken into a furin-like protease in the trans-Golgi network at two sites, resulting in two disulfide-linked subunits, F1 and F2, and releasing an undisclosed 27-amino-acid (aa) fragment, p27 [10,11].…”

“…Trimerized and completely cleaved F endorses a fusion-competent, metastable prefusion state that is located on the surface of infected cells in which it is inserted into developing infectious virons [10].…”