In recent studies we have focused on (1) mechanisms of influenza hemagglutinin mediated fusion and (2) late stages of cell-to-cell fusion. (1) The molecular mechanisms of fusion of lipid bilayers in remodeling of intracellular membranes, in entry of enveloped viruses and in cell-to-cell fusion in development remain to be clarified even for the best characterized examples of biological fusion. In one of our recent projects we have focused on fusion mediated by the influenza virus hemagglutinin (HA) that delivers viral RNA into cytosol by fusion between the viral envelope and the endosomal membrane. In the initial conformation of HA, its fusogenic subunit, the transmembrane protein HA2, is locked in a metastable conformation by the receptor-binding HA1 subunit of HA. Acidification in the endosome triggers HA2 refolding towards the final lowest energy conformation. Is the fusion process driven by this final conformation or, as often suggested, by the energy released by protein restructuring? To address this question we explored structural properties as well as the fusogenic activity of the full-sized trimeric HA2(1-185) (referred to as HA2*) that presents the final conformation of the HA2 ectodomain. We found HA2* to mediate fusion between lipid bilayers and between biological membranes. Two mutations known to inhibit HA-mediated fusion strongly inhibit the fusogenic activity of HA2*. At surface densities similar to those of HA in the influenza virus particle, HA2* forms small fusion pores but does not expand them. Interestingly, HA2* mediates lipid and content mixing only after application of acidic pH. The pH dependence of the HA2*-mediated fusion is in the range of those observed for other pH-dependent functions of the full-sized HA of the X-31 strain of influenza virus. The ability of HA2* to mediate low pH-dependent fusion suggests that there must be functionally important low pH-dependent interactions between the final conformations of HA2 subunits and/or between these conformations and the membranes. Indeed, acidic pH is known to affect conformation and self-association of membrane-inserted synthetic peptides mimicking the HA2 N-terminal fusion peptide domains of HA. Specific mechanisms by which the complete ectodomain HA2 fuses both model and biological membranes as well as the role of the fusogenic properties of the final conformation of HA2 subunit in the context of fusion mediated by full- sized HA, remain to be established. However our findings suggest that formation of the hairpin conformation of HA2, often referred to as a post-fusion conformation, does not signify the end of functionally important HA- and membrane- restructuring but rather represent the beginning of the key fusion stage. Restructuring of protein fusogens into a hairpin-like final structure under fusion conditions is a strikingly conserved mechanistic motif shared by very diverse viruses. Thus the question whether these hairpin conformations represent discharged post-fusion forms of the proteins or their functional fusogenic forms, is not limited solely to fusion mediated by HA. (2) Early stages of cell-to-cell fusion in developmental processes such as fertilization, muscle and bone formation and in pathological processes including viral infections and carcinogenesis culminate in the opening of nanometer-sized fusion pores that connect the volumes of two cells. The mechanisms that drive subsequent expansion of the fusion pore(s) to a micrometer-sized lumen that allows complete coalescence of cytoplasms are poorly understood. The membrane bilayer at the edge of the pore growing within the tight contact zone remains strongly curved and, hence, accumulates the elastic energy of bending. The degree of membrane bending at the pore rim is similar to that of intracellular membrane structures such as membrane tubules and endocytic vesicles. We have hypothesized that the cytosolic proteins involved in the cell-controlled bending of intracellular membranes accumulate at the fusion pore rim, lower its energy and, thus, promote pore expansion and syncytium formation. We explored whether changes in the activity of the proteins that generate these compartments affect cell fusion initiated by protein fusogens of influenza virus and baculovirus. We raised the intracellular concentration of curvature generating proteins in cells by either expressing or microinjecting the ENTH domain of Epsin or by expressing GRAF1 BAR domain or FCHo2 F-BAR domain. Each of these treatments promoted syncytium formation. Cell fusion extents were also influenced by treatments targeting the function of another curvature generating protein, dynamin. Cell membrane permeable inhibitors of dynamin GTPase blocked expansion of fusion pores and dominant-negative mutants of dynamin influenced the syncytium formation extents. We also found that syncytium formation is inhibited by reagents lowering the content and accessibility of phosphatidylinositol(4,5)bisphosphate, an important regulator of intracellular membrane remodeling. Our findings indicate that fusion pore expansion at late stages of cell-to-cell fusion is mediated, directly or indirectly, by intracellular membrane-shaping proteins. However a number of important questions remain open. To start with, the mechanisms underlying this dependence are yet to be clarified. Curvature-generating proteins can directly facilitate fusion pore expansion by accumulating at the pore edge and lowering its energy. These proteins can also promote vesiculation of the membrane junction at the edge of the fusion pores. Better understanding of the mechanism and cell machinery responsible for driving fusion pore expansion in cell-to-cell fusion will bring about new ways of controlling fusion in development and in pathological conditions. The dependence of fusion pore expansion on cell machinery can also be of importance for understanding why transient nanotubular connections between plasma membranes of some cells do not expand to yield multinucleated cells. Furthermore our work emphasizes an interesting overlap between proteins controlling late stages of cell-to-cell fusion and proteins that drive oppositely directed process of membrane remodeling, fission of one cell membrane into two. Curvature-generating proteins that we found to influence syncytium formation are essential components of different endocytic pathways. Further elucidation of the overlap between the protein players involved in the processes that unite and divide biological membranes is important for finding shared mechanistic principles underlying fusion and fission.

Project Start
Project End
Budget Start
Budget End
Support Year
20
Fiscal Year
2011
Total Cost
$1,450,984
Indirect Cost
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Zaitseva, Elena; Zaitsev, Eugene; Melikov, Kamran et al. (2017) Fusion Stage of HIV-1 Entry Depends on Virus-Induced Cell Surface Exposure of Phosphatidylserine. Cell Host Microbe 22:99-110.e7
Valansi, Clari; Moi, David; Leikina, Evgenia et al. (2017) Arabidopsis HAP2/GCS1 is a gamete fusion protein homologous to somatic and viral fusogens. J Cell Biol 216:571-581
Zicari, Sonia; Arakelyan, Anush; Fitzgerald, Wendy et al. (2016) Evaluation of the maturation of individual Dengue virions with flow virometry. Virology 488:20-7
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Uygur, Berna; Abramo, Katrina; Leikina, Evgenia et al. (2015) SLUG is a direct transcriptional repressor of PTEN tumor suppressor. Prostate 75:907-16
Kozlov, Michael M; Chernomordik, Leonid V (2015) Membrane tension and membrane fusion. Curr Opin Struct Biol 33:61-7
Melikov, Kamran; Hara, Ann; Yamoah, Kwabena et al. (2015) Efficient entry of cell-penetrating peptide nona-arginine into adherent cells involves a transient increase in intracellular calcium. Biochem J :
Leikina, Evgenia; Defour, Aurelia; Melikov, Kamran et al. (2015) Annexin A1 Deficiency does not Affect Myofiber Repair but Delays Regeneration of Injured Muscles. Sci Rep 5:18246
Bricceno, Katherine V; Martinez, Tara; Leikina, Evgenia et al. (2014) Survival motor neuron protein deficiency impairs myotube formation by altering myogenic gene expression and focal adhesion dynamics. Hum Mol Genet 23:4745-57
Kozlov, Michael M; Campelo, Felix; Liska, Nicole et al. (2014) Mechanisms shaping cell membranes. Curr Opin Cell Biol 29:53-60

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