Identification of Residues in the Human Respiratory Syncytial Virus Fusion Protein That Modulate Fusion Activity and Pathogenesis

Abstract

Human respiratory syncytial virus (RSV) lower respiratory tract infection can result in inflammation and mucus plugging of airways. RSV strain A2-line19F induces relatively high viral load and mucus in mice. The line 19 fusion (F) protein harbors five unique residues compared to the non-mucus-inducing strains A2 and Long, at positions 79, 191, 357, 371, and 557. We hypothesized that differential fusion activity is a determinant of pathogenesis. In a cell-cell fusion assay, line 19 F was more fusogenic than Long F. We changed the residues unique to line 19 F to the corresponding residues in Long F and identified residues 79 and 191 together as responsible for high fusion activity. Surprisingly, mutation of residues 357 or 357 with 371 resulted in gain of fusion activity. Thus, we generated RSV F mutants with a range of defined fusion activity and engineered these into recombinant viruses. We found a clear, positive correlation between fusion activity and early viral load in mice; however, we did not detect a correlation between viral loads and levels of airway mucin expression. The F mutant with the highest fusion activity, A2-line19F-K357T/Y371N, induced high viral loads, severe lung histopathology, and weight loss but did not induce high levels of airway mucin expression. We defined residues 79/191 as critical for line 19 F fusion activity and 357/371 as playing a role in A2-line19F mucus induction. Defining the molecular basis of the role of RSV F in pathogenesis may aid vaccine and therapeutic strategies aimed at this protein. IMPORTANCEHuman respiratory syncytial virus (RSV) is the most important lower respiratory tract pathogen of infants for which there is no vaccine. Elucidating mechanisms of RSV pathogenesis is important for rational vaccine and drug design. We defined specific amino acids in the fusion (F) protein of RSV strain line 19 critical for fusion activity and elucidated a correlation between fusion activity and viral load in mice. Further, we identified two distinct amino acids in F as contributing to the mucogenic phenotype of the A2-line19F virus. Taken together, these results illustrate a role for RSV F in virulence.H uman respiratory syncytial virus (hRSV according to the International Committee on Taxonomy of Viruses; often abbreviated RSV) is a negative-sense, nonsegmented, and singlestranded RNA virus in the Paramyxoviridae family. RSV is the most important viral lower respiratory tract pathogen for infants, causing 16 times more hospitalizations than influenza virus in children under 1 year old (1). There is currently no vaccine licensed to prevent RSV infections; there is also a need for additional therapies, and further knowledge of RSV pathogenesis will enhance efforts toward these goals.Strains of RSV exhibit differential pathogenesis in the BALB/c mouse model of RSV disease (2-4). A strain called line 19 was originally isolated in 1967 and then passaged in primary chick kidney cells, as well as primary chick lung cells, prior to 72 passages in human MRC-5 fibroblast cells (...

A Recombinant Respiratory Syncytial Virus Vaccine Candidate Attenuated by a Low-Fusion F Protein Is Immunogenic and Protective against Challenge in Cotton Rats

“…A dual-split-protein (DSP) cell-to-cell fusion assay was used to quantify fusion activity of expressed RSV F proteins as described previously (23)(24)(25). The consensus BAF gene was obtained in mammalian codon optimized form from GeneArt and cloned into pcDNA3.1.…”

Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication

“…In viral infections affecting the upper or lower respiratory tracts, such as influenza viruses and respiratory syncytial virus (RSV), the increased production of mucus, which is largely composed of mucins, is commonly induced in the epithelial surfaces of the respiratory tract (11,12). Recent studies have revealed that mucins are upregulated in epithelial cells soon after infection by respiratory viruses, such as influenza A virus (IAV), both in vivo and in vitro.…”

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

“…This raises the interesting question of the tolerance of fusion protein mutations in the context of infectious virus, especially in relation to our fusion-increasing mutations. This study and others have seen the fusogenicity of transfected F mutations mimic the same phenotype when they are part of an infectious virus (32,33). Even though many of our mutations show an increase in the fusion phenotype, the amino acid sequence of HRC is genetically stable, suggesting that the area does not tolerate change.…”

“…However, circulating virus is faced with the challenge of withholding transition until an appropriate host cell membrane is available. Correlation has been found between an increase in fusion activity and higher viral load and pathogenesis in mice for both RSV (33) and other paramyxoviruses (34). Thus, circulating virus faces competing selective pressures of balancing the infectious half-life of transmitted virions and fusion capacity.…”