Objective: To investigate the effect of the leucine zipper-like motif between HRA and HRB of the human parainfluenza virus 3 fusion protein on fusion activity. Methods: Site-directed mutagenesis was utilized to substitute the heptadic residues at 257, 264, 271, 278, 285, 292, and 299 in this motif with alanine. Additionally, 3 middle heptadic leucine residues at 271, 278, and 285 were replaced with alanine singly or in combination. A vaccinia virus-T7 RNA polymerase transient expression system was employed to express the wild-type or mutated fusion (F) proteins. Three different types of membrane fusion assays were performed to analyze the fusogenic activity, fluorescence-activated cell sorting (FACS) analysis was executed to examine the cell surface expression level, and a coimmunoprecipitation assay was conducted to probe the hemagglutinin-neuraminidase (HN)-F interaction at the cell surface. Results: All of the substitutions in this motif exhibited diminished or even lost fusion activity in all stages of fusion, although they all had no effect on cell surface expression. In the coimmunoprecipitation assay, all mutants resulted in decreased detection of the HN-F complexes compared with that of the wild-type F protein. Conclusions: This motif has an important influence on fusion activity, and its integrality is indispensable for membrane fusion.

Keywords:
Human parainfluenza virus 3, Fusion protein, Leucine zipper-like motif, Mutations, Membrane fusion
1.
Liu WK, Liu Q, Chen DH, Liang HX, Chen XK, Huang WB, Qin S, Yang ZF, Zhou R: Epidemiology and clinical presentation of the four human parainfluenza virus types. BMC Infect Dis 2013;13:28.
2.
Weinberg GA, Hall CB, Iwane MK, Poehling KA, Edwards KM, Griffin MR, Staat MA, Curns AT, Erdman DD, Szilagyi PG: Parainfluenza virus infection of young children: estimates of the population-based burden of hospitalization. J Pediatr 2009;154:694-699.
3.
Fry AM, Curns AT, Harbour K, Hutwagner L, Holman RC, Anderson LJ: Seasonal trends of human parainfluenza viral infections: United States, 1990-2004. Clin Infect Dis 2006;43:1016-1022.
4.
Stegmann T, Doms RW, Helenius A: Protein-mediated membrane fusion. Annu Rev Biophys Biophys Chem 1989;18:187-211.
5.
Lamb RA: Paramyxovirus fusion: a hypothesis for changes. Virology 1993;197:1-11.
6.
Crennell S, Takimoto T, Portner A, Taylor G: Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase. Nat Struct Biol 2000;7:1068-1074.
7.
Lawrence MC, Borg NA, Streltsov VA, Pilling PA, Epa VC, Varghese JN, McKimm-Breschkin JL, Colman PM: Structure of the haemagglutinin-neuraminidase from human parainfluenza virus type III. J Mol Biol 2004;335:1343-1357.
8.
Choppin PW, Scheid A: The role of viral glycoproteins in adsorption, penetration, and pathogenicity of viruses. Rev Infect Dis 1980;2:40-61.
9.
Lamb RA, Paterson RG, Jardetzky TS: Paramyxovirus membrane fusion: lessons from the F and HN atomic structures. Virology 2006;344:30-37.
10.
Scheid A, Choppin PW: Identification of biological activities of paramyxovirus glycoproteins: activation of cell fusion, hemolysis, and infectivity of proteolytic cleavage of an inactive precursor protein of Sendai virus. Virology 1974;57:475-490.
11.
Epand RM: Fusion peptides and the mechanism of viral fusion. Biochim Biophys Acta 2003;1614:116-121.
12.
Baker KA, Dutch RE, Lamb RA, Jardetzky TS: Structural basis for paramyxovirus-mediated membrane fusion. Mol Cell 1999;3:309-319.
13.
Yu M, Wang E, Liu Y, Cao D, Jin N, Zhang CW, Bartlam M, Rao Z, Tien P, Gao GF: Six-helix bundle assembly and characterization of heptad repeat regions from the F protein of Newcastle disease virus. J Gen Virol 2002;83:623-629.
14.
Landschulz WH, Johnson PF, McKnight SL: The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 1988;240:1759-1764.
15.
O'Shea EK, Rutkowski R, Kim PS: Evidence that the leucine zipper is a coiled coil. Science 1989;243:538-542.
16.
Buckland R, Malvoisin E, Beauverger P, Wild F: A leucine zipper structure present in the measles virus fusion protein is not required for its tetramerization but is essential for fusion. J Gen Virol 1992;73:1703-1707.
17.
Reitter JN, Sergel T, Morrison TG: Mutational analysis of the leucine zipper motif in the Newcastle disease virus fusion protein. J Virol 1995;69:5995-6004.
18.
Ghosh JK, Ovadia M, Shai Y: A leucine zipper motif in the ectodomain of Sendai virus fusion protein assembles in solution and in membranes and specifically binds biologically-active peptides and the virus. Biochemistry 1997;36:15451-15462.
19.
Fuerst TR, Niles EG, Studier FW, Moss B: Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci USA 1986;83:8122-8126.
20.
Deng R, Wang Z, Mirza AM, Iorio RM: Localization of a domain on the paramyxovirus attachment protein required for the promotion of cellular fusion by its homologous fusion protein spike. Virology 1995;209:457-469.
21.
Mirza AM, Deng R, Iorio RM: Site-directed mutagenesis of a conserved hexapeptide in the paramyxovirus hemagglutinin-neuraminidase glycoprotein: effects on antigenic structure and function. J Virol 1994;68:5093-5099.
22.
Chu FL, Wen HL, Hou GH, Lin B, Zhang WQ, Song YY, Ren GJ, Sun CX, Li ZM, Wang Z: Role of N-linked glycosylation of the human parainfluenza virus type 3 hemagglutinin-neuraminidase protein. Virus Res 2013;174:137-147.
23.
Ren G, Wang Z, Wang G, Song Y, Yao P, Xu H, Wen H, Zhang W: Effects of heptad repeat regions of F protein on the specific membrane fusion in paramyxoviruses. Intervirology 2006;49:299-306.
24.
Sanchez-Felipe L, Villar E, Munoz-Barroso I: Entry of Newcastle disease virus into the host cell: role of acidic pH and endocytosis. Biochim Biophys Acta 2014;1838:300-309.
25.
Morris SJ, Sarkar DP, White JM, Blumenthal R: Kinetics of pH-dependent fusion between 3T3 fibroblasts expressing influenza hemagglutinin and red blood cells: measurement by dequenching of fluorescence. J Biol Chem 1989;264:3972-3978.
26.
Dutch RE, Joshi SB, Lamb RA: Membrane fusion promoted by increasing surface densities of the paramyxovirus F and HN proteins: comparison of fusion reactions mediated by simian virus 5 F, human parainfluenza virus type 3 F, and influenza virus HA. J Virol 1998;72:7745-7753.
27.
Bagai S, Lamb RA: Quantitative measurement of paramyxovirus fusion: differences in requirements of glycoproteins between simian virus 5 and human parainfluenza virus 3 or Newcastle disease virus. J Virol 1995;69:6712-6719.
28.
Apte-Sengupta S, Negi S, Leonard VH, Oezguen N, Navaratnarajah CK, Braun W, Cattaneo R: Base of the measles virus fusion trimer head receives the signal that triggers membrane fusion. J Biol Chem 2012;287:33026-33035.
29.
McGinnes LW, Morrison TG: Inhibition of receptor binding stabilizes Newcastle disease virus HN and F protein-containing complexes. J Virol 2006;80:2894-2903.
30.
Gravel KA, McGinnes LW, Reitter J, Morrison TG: The transmembrane domain sequence affects the structure and function of the Newcastle disease virus fusion protein. J Virol 2011;85:3486-3497.
31.
Earp LJ, Delos SE, Park HE, White JM: The many mechanisms of viral membrane fusion proteins. Curr Top Microbiol Immunol 2005;285:25-66.
32.
Hernandez LD, Hoffman LR, Wolfsberg TG, White JM: Virus-cell and cell-cell fusion. Annu Rev Cell Dev Biol 1996;12:627-661.
33.
Buckland R, Wild F: Leucine zipper motif extends. Nature 1989;338:547.
34.
Lambert DM, Barney S, Lambert AL, Guthrie K, Medinas R, Davis DE, Bucy T, Erickson J, Merutka G, Petteway SR Jr: Peptides from conserved regions of paramyxovirus fusion (F) proteins are potent inhibitors of viral fusion. Proc Natl Acad Sci USA 1996;93:2186-2191.
35.
Luo Z, Matthews AM, Weiss SR: Amino acid substitutions within the leucine zipper domain of the murine coronavirus spike protein cause defects in oligomerization and the ability to induce cell-to-cell fusion. J Virol 1999;73:8152-8159.
36.
Jain S, McGinnes LW, Morrison TG: Thiol/disulfide exchange is required for membrane fusion directed by the Newcastle disease virus fusion protein. J Virol 2007;81:2328-2339.
37.
Kemble GW, Danieli T, White JM: Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion. Cell 1994;76:383-391.
38.
West DS, Sheehan MS, Segeleon PK, Dutch RE: Role of the simian virus 5 fusion protein N-terminal coiled-coil domain in folding and promotion of membrane fusion. J Virol 2005;79:1543-1551.
39.
Hu XL, Ray R, Compans RW: Functional interactions between the fusion protein and hemagglutinin-neuraminidase of human parainfluenza viruses. J Virol 1992;66:1528-1534.
40.
Melanson VR, Iorio RM: Amino acid substitutions in the F-specific domain in the stalk of the Newcastle disease virus HN protein modulate fusion and interfere with its interaction with the F protein. J Virol 2004;78:13053-13061.
41.
Tsurudome M, Ito M, Nishio M, Nakahashi M, Kawano M, Komada H, Nosaka T, Ito Y: Identification of domains on the fusion (F) protein trimer that influence the hemagglutinin-neuraminidase specificity of the F protein in mediating cell-cell fusion. J Virol 2011;85:3153-3161.
42.
Tsurudome M, Ito M, Nishio M, Kawano M, Okamoto K, Kusagawa S, Komada H, Ito Y: Identification of regions on the fusion protein of human parainfluenza virus type 2 which are required for haemagglutinin-neuraminidase proteins to promote cell fusion. J Gen Virol 1998;79:279-289.
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