Our long-term goal is to understand how proteins remodel membrane lipid bilayers in important cell biology processes. In recent studies we have focused on mechanisms of fusion stage of dengue virus (DEN) infection and escape of cationic cell-penetrating peptides (CPP) from endosomes. DEN, the most prevalent mosquito-borne virus world-wide, is endemic in more than 100 countries with an estimated 100 million cases of DEN infections per year. 90% of 500,000 hospitalizations annually are children. Due to climate change and the increased mobility of people across national borders, this infectious tropical disease is an ever-growing global threat to health and the economy. Dengue hemorrhagic fever is a leading cause of death among children in some Asian countries. Currently there are neither vaccines nor effective therapies for DEN infections. As many other viruses, cell bound and then internalized DEN delivers its genome into cells by fusion between viral membrane and membrane of acidified endosome. Fusion stage of viral entry is an important target for antivirals (including HIV-1 fusion inhibitor enfuvirtide). However screening of potential antivirals of this class requires application of fusion assays such as virus-mediated cell fusion or virus fusion to protein free model membranes (liposomes). Surprisingly, development of such assays for DEN has been unsuccessful. Also, surprisingly, DEN has been reported to fuse only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors required for DEN fusion that time fusion to virion entry into late endosomes and had been lacking in earlier attempts to achieve DEN fusion to plasma membrane of mammalian cells and liposomes? In our study we found that DEN utilizes bis(monoacylglycero)phosphate, a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery. Effective virus fusion to plasma- and intracellular- membranes, as well as to protein-free liposomes, requires the target membrane to contain anionic lipids such as bis(monoacylglycero)phosphate and phosphatidylserine. Anionic lipids act downstream of low-pH-dependent fusion stages and promote the advance from the earliest hemifusion intermediates to the fusion pore opening. To reach anionic lipid-enriched late endosomes, DEN travels through acidified early endosomes but we found that low pH-dependent loss of fusogenic properties of DEN is relatively slow in the presence of anionic lipid-free target membranes. We propose that anionic lipid-dependence of DEN fusion machinery protects it against premature irreversible restructuring and inactivation and ensures viral fusion in late endosomes, where the virus encounters anionic lipids for the first time during entry. As a result, DEN effectively delivers viral RNA to its translation/replication sites. Identification of the essential cofactor of DEN fusion machinery allowed us to develop an arsenal of fusion assays (virus-liposome fusion, virus-cell fusion and DEN intracellular fusion). The assays developed in this study to directly characterize DEN fusion will hopefully help in developing antivirals including those targeting DEN interactions with anionic lipids to block or prematurely activate viral fusion machinery. Furthermore, interactions between protein fusogen of DEN protein E and anionic lipids may have implications for the pathogenesis of dengue hemorrhagic fever, which is characterized by activation of endothelial cells and extracellular exposure of anionic lipids. In another project we explored mechanisms of delivery cationic cell penetrating peptides into cytosol using model lipid bilayers. CPPs such as TAT peptide and other arginine-rich peptides are a promising vehicle for the delivery of macromolecular drugs. While many studies indicate that CPPs enter cells by endocytosis, mechanisms by which they cross endosomal membranes remain elusive. Moreover some papers indicate that endosomal escape is a limiting factor in endocytosis dependent delivery of functionally active CPP-cargo conjugates into cytosol and nucleus. Thus understanding the mechanism of the endosomal escape is of great practical importance. In our recent work we have proposed a new model for the delivery of cationic CPPs and conjugated cargo into cytosol. This model is based on both characteristic multivesicular morphology of late endosomes and the fact that intraluminal vesicles are highly enriched with late endosome specific anionic lipid bis(monoacylglycero)phosphate. We hypothesize that cationic peptides induce leaky fusion (i.e., fusion associated with membrane permeabilization) between intraluminal vesicles, resulting in delivery of the peptide and cargo molecules into intraluminal vesicles. Subsequent back fusion of intraluminal vesicle with the limiting membrane of late endosomes releases peptide and cargo into cell cytosol. To test this model we focused on interactions of TAT peptide with model protein-free lipid bilayers liposomes of various lipid compositions including those mimicking lipid bilayers of plasma membrane and late endosomes. We report that TAT peptide induces leakage of encapsulated probes and translocates across bis(monoacylglycero)phosphate-enriched bilayers mimicking lipid composition of intraluminal vesicles of late endosome but not across membranes mimicking lipid composition of plasma membranes. TAT-induced leakage and translocation are linked to membrane fusion and inhibited by fusion inhibitors. These results substantiate the proposed model of endosomal escape and suggest that membrane fusion known to be an important stage of intracellular delivery for membrane-enclosed macromolecules such as viral nucleic acids may be also involved in delivery of membrane-free macromolecules.

Project Start
Project End
Budget Start
Budget End
Support Year
19
Fiscal Year
2010
Total Cost
$1,143,528
Indirect Cost
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State
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Verma, Santosh K; Chernomordik, Leonid V; Melikov, Kamran (2018) An improved metrics for osteoclast multinucleation. Sci Rep 8:1768
Verma, Santosh K; Leikina, Evgenia; Melikov, Kamran et al. (2018) Cell-surface phosphatidylserine regulates osteoclast precursor fusion. J Biol Chem 293:254-270
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
Gamage, Dilani G; Leikina, Eugenia; Quinn, Malgorzata E et al. (2017) Insights into the localization and function of myomaker during myoblast fusion. J Biol Chem 292:17272-17289
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
Uygur, Berna; Abramo, Katrina; Leikina, Evgenia et al. (2015) SLUG is a direct transcriptional repressor of PTEN tumor suppressor. Prostate 75:907-16
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 :
Kozlov, Michael M; Chernomordik, Leonid V (2015) Membrane tension and membrane fusion. Curr Opin Struct Biol 33:61-7
Chernomordik, Leonid V; Kozlov, Michael M (2015) Myoblast fusion: playing hard to get. Dev Cell 32:529-30

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