Asterisks indicate a significant difference as measured by Student’s test ( 0

Asterisks indicate a significant difference as measured by Student’s test ( 0.05). residues that were neither located at a or d positions in the heptad repeat nor conserved among the paramyxoviruses were key regulators of the folding and fusion activity of the F protein, showing that residues not expected to be important in coiled-coil formation may play important functions in regulating membrane fusion. Overall, the data support the hypothesis that regions in the F protein that undergo dramatic changes in secondary and tertiary structure between the prefusion and hairpin conformations regulate F protein expression and activation. Paramyxoviruses have evolved two surface glycoproteins that cause membrane fusion during viral entry: a receptor-binding (HN, H, or G) protein and a fusion (F) protein (31). During viral entry, the receptor-binding protein attaches to its cellular receptor and then transduces a signal to Rabbit Polyclonal to SHP-1 the F protein to initiate membrane fusion (12, 22, 40, 46, 57). As a class I viral fusion protein, the paramyxovirus F protein is usually synthesized as a single precursor protein (F0), folded as a homotrimer, glycosylated, and cleaved into an active form consisting of a small amino-terminal subunit (F2) and a larger carboxy-terminal subunit (F1) (31). The ectodomain of the F1 subunit contains a hydrophobic fusion peptide at its amino terminus and two 4-3 heptad repeat regions, HRA and HRB (Fig. ?(Fig.1A).1A). Upon activation, the F protein is thought to insert its hydrophobic fusion peptide into the target membrane and form a coiled-coil hairpin structure with its HRA and HRB regions (32, 48) in order to actively drive membrane fusion (35, 46). Open in a separate windows FIG. 1. The paramyxovirus F protein. (A) Domain structure of the F protein. HRA and HRB are shown in red and blue, respectively. The signal peptide (SP), fusion peptide (FP), transmembrane (TM), and cytoplasmic tail (CT) regions are also labeled. Structural domains DI, DII, and DIII are represented by solid lines. (B) Sequence Alogliptin alignment of HRA regions of SeV, Nipah computer virus, hPIV3, PIV5, and NDV. Identical residues are highlighted in red, and comparable residues are highlighted in yellow. The black box identifies the sequence of the 10 conserved residues that were mutated in this study. The secondary structures of the region in the prefusion (native) and hairpin (final) conformations of the F protein are shown, with bars representing Alogliptin -helices and arrows representing -strands. Heptad repeat a and d residues are shown underneath the sequence alignment. The underlines correspond to a stutter in the heptad repeat (1). (C) Structure of the PIV5 F protein ectodomain in its uncleaved, prefusion form (64). (D) Structure of the hPIV3 F protein ectodomain in its hairpin form (63). In both panels C and D, HRA is shown in red and HRB in blue. The insets show the structures formed by one monomer of HRA, and the boxes identify the residues investigated in this study. Panels C and D were rendered in MOLMOL (30). Peptides derived from the HRA and HRB regions of the paramyxovirus F protein inhibit membrane fusion and computer virus replication (3, 33, 39, 46, 62) by mechanisms similar to those of HR-derived peptides of human immunodeficiency computer virus (HIV) type 1 gp41 (6, 21, 25). HRA-derived peptides are thought to bind HRB in an early intermediate of the F protein, and HRB-derived peptides bind to HRA in a prehairpin intermediate (46, 47). In both cases, the binding of an HR-derived peptide to its complementary HR region in the F protein prevents formation of the hairpin that is needed to drive membrane fusion (35, 46). HRB-derived peptides are shorter, more soluble, and approximately 1,000 times as potent as HRA-derived peptides (19). Therefore, antiviral strategies often use HRB-derived peptides and small molecules to target the HRA coiled coil in the prehairpin intermediate (14). HRB-derived peptides usually inhibit membrane fusion mediated only by the F protein from which they are derived (31). However, inhibition of closely related F proteins has been exhibited (3), and an HRB-derived peptide from human parainfluenza computer virus type 3 (hPIV3) has been shown to inhibit Hendra computer virus fusion (39). The mechanism of cross-species inhibition is usually unresolved but may involve the topography of the.G. Sendai computer virus F protein a highly conserved 10-residue sequence in HRA that undergoes major structural changes during protein refolding. Nine of the 15 mutations studied caused significant defects in F protein expression, processing, and fusogenicity. Conversely, the remaining six mutations enhanced the fusogenicity of the F protein, most likely by helping spring the HRA coil. Two of the residues that were neither located at a or d positions in the heptad repeat nor conserved among the paramyxoviruses were key regulators of the folding and fusion activity of the F protein, showing that residues not expected to be important in coiled-coil formation may play important functions in regulating membrane fusion. Overall, the data support the hypothesis that regions in the F protein that undergo dramatic changes in secondary and tertiary structure between the prefusion and hairpin conformations regulate F protein expression and activation. Paramyxoviruses have evolved two surface glycoproteins that cause membrane fusion during viral entry: a receptor-binding (HN, H, or G) protein and a fusion (F) protein (31). During viral entry, the receptor-binding protein attaches to its cellular receptor and then transduces a signal to the F protein to initiate membrane fusion (12, 22, 40, 46, 57). As a class I viral fusion protein, the paramyxovirus F protein is usually synthesized as a single precursor protein (F0), folded as a homotrimer, glycosylated, and cleaved into an active form consisting of a small amino-terminal subunit (F2) and a larger carboxy-terminal subunit (F1) (31). The ectodomain of the F1 subunit contains a hydrophobic fusion peptide at its amino terminus and two 4-3 heptad repeat regions, HRA and Alogliptin HRB (Fig. ?(Fig.1A).1A). Upon activation, the F protein is thought to insert its hydrophobic fusion peptide into the target membrane and form a coiled-coil hairpin structure with its HRA and HRB regions (32, 48) in order to positively travel membrane fusion (35, 46). Open up in another windowpane FIG. 1. The paramyxovirus F proteins. (A) Domain framework from the F proteins. HRA and HRB are demonstrated in reddish colored and blue, respectively. The sign peptide (SP), fusion peptide (FP), transmembrane (TM), and cytoplasmic tail (CT) areas are also tagged. Structural domains DI, DII, and DIII are displayed by solid lines. (B) Series positioning of HRA parts of SeV, Nipah disease, hPIV3, PIV5, and NDV. Identical residues are highlighted in reddish colored, and identical residues are highlighted in yellowish. The black package identifies the series from the 10 conserved residues which were mutated with this research. The secondary constructions of the spot in the prefusion (indigenous) and hairpin (last) conformations from the F proteins are demonstrated, with pubs representing -helices and arrows representing -strands. Heptad do it again a and d residues are demonstrated underneath the series positioning. The underlines match a stutter in the heptad do it again (1). (C) Framework from the PIV5 F proteins ectodomain in its uncleaved, prefusion type (64). (D) Framework from the hPIV3 F proteins ectodomain in its hairpin type (63). In both sections C and D, HRA can be shown in reddish colored and HRB in blue. The insets display the structures shaped by one monomer of HRA, as well as the containers determine the residues looked into in this research. Sections C and D had been rendered in MOLMOL (30). Peptides produced from the HRA and HRB parts of the paramyxovirus F proteins inhibit membrane fusion and disease replication (3, 33, 39, 46, 62) by systems just like those of HR-derived peptides of human being immunodeficiency disease (HIV) type 1 gp41 (6, 21, 25). HRA-derived peptides are believed to bind HRB within an early intermediate from the F proteins, and HRB-derived peptides bind to HRA inside a prehairpin intermediate (46, 47). In both instances, the binding of the HR-derived peptide to its complementary HR area in the F proteins prevents formation from the hairpin that’s needed Alogliptin to travel membrane fusion (35,.