Potential Electrostatic Interactions in Multiple Regions Affect Human Metapneumovirus F-Mediated Membrane Fusion

Abstract

<jats:p>The recently identified human metapneumovirus (HMPV) is a worldwide respiratory virus affecting all age groups and causing pneumonia and bronchiolitis in severe cases. Despite its clinical significance, no specific antiviral agents have been approved for treatment of HMPV infection. Unlike the case for most paramyxoviruses, the fusion proteins (F) of a number of strains, including the clinical isolate CAN97-83, can be triggered by low pH. We recently reported that residue H435 in the HRB linker domain acts as a pH sensor for HMPV CAN97-83 F, likely through electrostatic repulsion forces between a protonated H435 and its surrounding basic residues, K295, R396, and K438, at low pH. Through site-directed mutagenesis, we demonstrated that a positive charge at position 435 is required but not sufficient for F-mediated membrane fusion. Arginine or lysine substitution at position 435 resulted in a hyperfusogenic F protein, while replacement with aspartate or glutamate abolished fusion activity. Studies with recombinant viruses carrying mutations in this region confirmed its importance. Furthermore, a second region within the F<jats:sub>2</jats:sub>domain identified as being rich in charged residues was found to modulate fusion activity of HMPV F. Loss of charge at residues E51, D54, and E56 altered local folding and overall stability of the F protein, with dramatic consequences for fusion activity. As a whole, these studies implicate charged residues and potential electrostatic interactions in function, pH sensing, and overall stability of HMPV F.</jats:p>

Breaking In: Human Metapneumovirus Fusion and Entry

“…Interestingly, a predicted cluster of positively charged residues in this same region of the AMPV-A F protein (a distantly related pneumovirus) are important for fusion activity at neutral pH [75]. These studies suggest that the His residue at position 435 may serve as a low-pH sensor, and one model proposed that protonation of the His residue at low pH induces localized electrostatic repulsion that leads to the destabilization of F and fusion triggering [73,76]. Alternatively, electrostatic repulsion among the basic residues induced by receptor binding could destabilize the HRB linker domain and trigger fusion [75].…”

“…Acidic residues Glu 51 , Asp 54 , and Glu 56 (Figure 3B, yellow spheres) are important for low-pH triggered F-mediated cell-cell fusion, but also appear to be critical for protein stability [76]. These residues are clustered in a charged region present in the F2 subunit of F in close proximity to two basic residues (Figure 3B, brown spheres) [76].…”

“…Acidic residues Glu 51 , Asp 54 , and Glu 56 (Figure 3B, yellow spheres) are important for low-pH triggered F-mediated cell-cell fusion, but also appear to be critical for protein stability [76]. These residues are clustered in a charged region present in the F2 subunit of F in close proximity to two basic residues (Figure 3B, brown spheres) [76]. The specific acidic residues in the F2 subunit and basic residues in the HRA domain are highly conserved in all HMPV strains; therefore, others have predicted that salt bridges and/or electrostatic interactions in this region may contribute to F triggering during HMPV fusion [76].…”

“…These residues are clustered in a charged region present in the F2 subunit of F in close proximity to two basic residues (Figure 3B, brown spheres) [76]. The specific acidic residues in the F2 subunit and basic residues in the HRA domain are highly conserved in all HMPV strains; therefore, others have predicted that salt bridges and/or electrostatic interactions in this region may contribute to F triggering during HMPV fusion [76]. This hypothesis is supported by another study that indicated that the F2 subunit from AMPV-C could confer a hyperfusogenic phenotype to HMPV F1 [62].…”

The Human Metapneumovirus Small Hydrophobic Protein Has Properties Consistent with Those of a Viroporin and Can Modulate Viral Fusogenic Activity

“…At 18 h posttransfection, the cells were starved, metabolically labeled with Tran 35 S-label (100 Ci/ml; PerkinElmer, Waltham, MA), and either lysed or subjected to surface biotinylation prior to lysis, as previously described (33). Antipeptide serum to HMPV F or G (32) or the monoclonal Ab 12CA5 to the HA tag (Roche) was used to immunoprecipitate the desired proteins as previously described (34). Biotin-labeled protein was pulled down with immobilized streptavidin (Thermo Scientific, Rockford, IL), as described previously (33).…”