The ability of membrane lipids to form continuous and closed bilayers is essential for many functions of biological membranes. However, many biological processes involve local and transient breaking of membrane continuity. In particular, protein-mediated membrane fusion is an important stage of exocytosis, protein trafficking, fertilization, and enveloped virus infection. We study the mechanisms by which specialized proteins reshape and remodel membranes and recently established the striking similarity of membrane rearrangement pathways in biological fusion driven by fusion proteins as diverse as alphavirus E1 and influenza hemagglutinin. In search of ways to control fusion reactions during viral entry, we concentrated on an unexplored stage of viral fusion. Virus-cell membrane contact is crowded with proteins (including fusion proteins and receptors) that cover both membranes. To allow tight bilayer contact, hemifusion, and fusion-pore opening, membrane proteins have to be displaced from the future fusion site. We were able to block displacement by cross-linking surface glycoproteins. Moreover, we found that several antiviral lectin components of the innate immunity block influenza virus infection by this novel displacement mechanism. We also studied the mechanisms by which cationic peptides deliver macromolecules into cells and explored the contributions of clathrin-dependent endocytosis and heparan sulfate receptors.
Similarity between membrane fusion pathways driven by the class II fusion protein of alphaviruses and class I influenza virus hemagglutinin
Zaitseva, Mittal, Chernomordik; in collaboration with Griffin
Recent studies on the diversity of fusion proteins have focused on proteins that mediate fusion by which enveloped viruses deliver their genome into host cells. Influenza and Sindbis viruses are among the best-studied prototypes of fusion machinery. For both viruses, fusion is triggered by acidification of the virus-containing endosome. While the final lowest-energy forms of Sindbis E1, influenza virus hemagglutinin (HA), and many other fusion proteins share an important motif, which takes the form of two sequences that interact with membranes, the fusion peptide and the transmembrane domain relocate to the same end of the rod-like molecule. However, HA and E1 differ radically in their initial structures and have come to represent two divergent classes of viral fusion proteins. Class I proteins (exemplified by HA and HIV env) are oriented perpendicularly to the envelope surface and feature alpha-helical coiled-coil domains. A highly conserved and fusion peptide sequence critical for fusion is located at or near the NH2-terminus of the fusion protein. Class II proteins (for instance, the E1 protein of Sindbis and Semliki Forest viruses and the E protein of flaviviruses) lie tangential to the virus membrane and have an internal rather than terminal fusion peptide. Class II proteins contain predominantly beta-strand secondary structures and are not predicted to form coiled coils.
We attempted to determine whether fusion pathways mediated by alphavirus E1 and HA, which exemplify classes II and I, differ and thus reflect the difference in their initial conformations, or whether they concur and thus reflect the similarity in their final conformations. We dissected the pathway of low pH–triggered E1-mediated cell-cell fusion by reducing the numbers of activated E1 proteins and blocking different fusion stages with specific inhibitors. The discovered progression from transient hemifusion to small and then expanding fusion pores upon an increase in the number of activated fusion proteins parallels that established for HA-mediated fusion. We conclude that proteins as different as E1 and HA drive fusion through strikingly similar membrane intermediates, with the most energy-intensive stages following rather than preceding hemifusion. Thus, fusion proteins of both classes (I and II) drive the entire fusion pathway rather than merely catalyze the merger of the contacting monolayers of two membranes.
Our finding that dissimilar viral fusion proteins catalyze a fusion-through-hemifusion pathway points to the universality of this mechanism of biological fusion. The results also support the hypothesis that the final hairpin structure shared by diverse viral fusion proteins and by proteins involved in intracellular fusion is more important for fusion than the initial metastable conformations of these proteins.
Leikina E, Mittal A, Cho MS, Melikov K, Kozlov MM, Chernomordik LV. Influenza hemagglutinins outside of the contact zone are necessary for fusion pore expansion. J Biol Chem 2004;279:26526-26532.
Zaitseva E, Mittal A, Griffin DE, Chernomordik LV. Class II fusion protein of alphaviruses drives membrane fusion through the same pathway as class I proteins. J Cell Biol 2005;169:167-177.
Inhibition of viral fusion and entry by carbohydrate-binding molecules that cross-link membrane glycoproteins
Leikina, Delanoe-Ayari, Melikov, Cho,3 Chen, Chernomordik; in collaboration with Lehrer, Loo, Wang Waring, Xie
For countless pathogenic viruses, including influenza virus and HIV, delivery of viral nucleic acid into a cell to initiate a productive infection requires fusion between the viral membrane envelope and the cell membrane. Membrane fusion mediated by viral envelope glycoproteins such as influenza virus HA or HIV env is an important target of antiviral agents. Viral entry can be inhibited by blocking binding between the virus and its receptors at the target cell surface; by interfering with the fusogenic activity of the viral fusion proteins; or by altering mechanical properties of membrane lipid bilayers to make the bilayers less fusogenic. Natural antiviral defenses are mediated by the adaptive and innate immune systems. Effector molecules of the adaptive system, such as antibodies, are typically highly pathogen-specific. In contrast, those of the innate system, such as defensins (antimicrobial peptides expressed by leukocytes and by epithelial cells that protect the host against microorganisms), generally have a broader spectrum that is not pathogen-specific.
Retrocyclin-2 (RC2), a circular octadecapeptide with three disulphide bonds, belongs to the family of theta-defensins. In humans, this peptide is encoded by pseudogenes whose transcripts are expressed in bone marrow. The ability of RC2 to inhibit HIV and herpes simplex virus infection makes it a promising candidate for therapeutic development. We found that RC2 inhibited viral entry by blocking membrane fusion. Three of our findings argue against a primary role of viral-specific interactions between RC2 and viral fusion proteins in this inhibition. First, RC2 inhibited fusion reactions driven not only by HA but also by the dissimilar fusion proteins of Sindbis virus and baculovirus. Second, RC2 could be applied only to the target membrane. Third, RC2 was effective after HA was restructured from an initial nonfusogenic conformation to a fusogenic one and was effective both before and after hemifusion. Using a fluorescence-recovery-after-photobleaching assay, we found that RC2, a multivalent lectin, immobilizes membrane glycoproteins. Thus, in contrast to fusion inhibitors that specifically target fusion proteins, receptors, or bilayers, RC2 prevented HA-mediated fusion by erecting a network of cross-linked and immobilized surface glycoproteins.
The lectin activity that underlies the fusion-inhibiting activity of RC2 has also been described for other important components of the innate immune system, including mannan-binding lectin (MBL) and human beta defensin-3 (HBD3). We found that human MBL and HBD3 have fusion- and membrane protein mobility–inhibiting activities similar to those observed for RC2. Cross-linking of surface glycoproteins by endogenous lectin-like host defense molecules can block an essential but hitherto unexplored stage of viral entry, namely, displacement of proteins from the prospective fusion site. Further exploration of this newly characterized antiviral mechanism and its place in innate immunity in vivo could lead to the development of new strategies and agents to prevent and treat viral infections.
Leikina E, Delanoe-Ayari H, Melikov K, Cho M-S, Chen A, Waring AJ, Wang W, Xie Y, Loo JA, Lehrer RI, Chernomordik LV. Carbohydrate-binding molecules inhibit viral fusion and entry by crosslinking membrane glycoproteins. Nat Immunol 2005;6:995-1001.
Involvement of clathrin-dependent endocytosis and heparan sulfate receptors in cellular uptake of TAT peptide
Melikov, Richard, Chernomordik; in collaboration with Brooks, Lebleu, Prevot
Accelerated by the completion of the human genome project, recent advances in the identification of new molecular therapy targets and disease-relevant proteins emphasize the importance for molecular therapy of high–molecular weight information-rich biomolecules, such as peptides, proteins, antisense DNA, and small interfering RNA. Cationic cell-penetrating peptides (CPP) such as the TAT peptide derived from the protein transduction domain of HIV TAT protein are widely considered to be a promising approach for delivery of proteins and nucleic acids into cells. Despite significant progress in the cytoplasmic and nuclear delivery of various cargo molecules using CPP, the underlying mechanisms remain under debate. We examined the mechanisms by which the TAT peptide enters living cells. We found TAT entry into several different primary cells and into many stable cell lines to be ATP- and temperature-dependent, indicating the involvement of endocytosis. Judging from the effects of specific inhibitors, we concluded that entry of the unconjugated TAT peptide into cells involves a clathrin-dependent endocytic pathway. In contrast, the caveolin-dependent pathway is not essential for the uptake of unconjugated TAT peptide, as evidenced by the efficient internalization of TAT in the presence of the known inhibitors of the raft/caveolin-dependent pathway, or for cells lacking or deficient in caveolin-1 expression.
To evaluate the role of heparan sulfates in the TAT uptake, we used mutant cells lacking surface heparan sulfate (pgs-A745 and pgs-D677 cell lines) and cells pretreated with heparinase III. Uptake inhibition under both conditions indicated the importance of heparan sulfate receptors for the uptake of TAT peptide in wild-type CHO cells. However, TAT internalization in the absence of heparan sulfate proteoglycans points to the existence of heparan sulfate–independent mechanisms of entry. Thus, whereas a significant part of TAT peptide uptake involves heparan sulfate receptors, efficient internalization of peptide is observed even in these receptors’ absence, indicating the involvement of other receptors.
To test whether endocytosed TAT peptide is targeted into acidified compartments, we took advantage of the fact that the fluorescence of fluorescein is significantly lower at the pH 5 to 6 typical for endosomes and lysosomes than at the pH 7.4 of cell culture media. We incubated HeLa cells with fluorescein-tagged TAT for 60 minutes followed by 30 minutes’ incubation in the presence or absence of monensin at 4°C and found that monensin significantly increases the cell-associated fluorescence. Thus, a significant fraction of the TAT peptide is delivered into acidic cellular compartments.
Our results along with those reported in the recent literature indicate that the uptake of CPPs and their cargo in different cells involves different types of endocytosis: clathrin-dependent endocytosis, raft/caveolin-dependent endocytosis, and macropinocytosis. CPPs dramatically increase the uptake of conjugated molecules through efficient binding to surface proteoglycans. Whether the increase in binding can ensure delivery of a sufficient amount of functionally active macromolecules into cytoplasm and nucleus or whether there is a specific mechanism by which CPPs facilitate escape of conjugated cargo from endosomes remains to be understood.
Melikov K, Chernomordik LV. Arginine rich cell penetrating peptides: from endosomal uptake to nuclear delivery. Cell Mol Life Sci[Epub ahead of print].
Richard JP, Melikov K, Brooks H, Prevot P, Lebleu B, Chernomordik LV. Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J Biol Chemistry 2005;280:15300-15306.
1Now at the CNRS, Université Montpellier 2, Montpellier, France
2Associate Professor, on sabbatical leave from Technion-Israel Institute of Technology, Haifa, Israel
3Myoung-Soon Cho, MS, Biologist, now at the Laboratory of Cell Biology, NHLBI, Bethesda, MD
Collaborators
Hilary Brooks,PhD, CNRS, Université Montpellier 2, Montpellier, France
Tamar Gattegno,MSc, Technion-Israel Institute of Technology, Haifa, Israel
Alessandra Gliozzi, PhD, Università di Genova, Genoa, Italy
Diane E. Griffin, PhD, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD
David H. Hall, PhD, Albert Einstein College of Medicine, Bronx, NY
Irina Kolotuev, MSc, Technion-Israel Institute of Technology, Haifa, Israel
Michael Kozlov, PhD, Tel Aviv University, Tel Aviv, Israel
Bernard Lebleu, PhD, CNRS, Université Montpellier 2, Montpellier, France
Robert I. Lehrer, MD, David Geffen School of Medicine at UCLA, Los Angeles, CA
Joseph A. Loo, PhD, University of California Los Angeles, Los Angeles, CA
Ken C.Q. Nguyen, MSc, Albert Einstein College of Medicine, Bronx, NY
Paul Prevot, PhD, CNRS, Université Montpellier 2, Montpellier, France
Gidi Shemer,PhD, Technion-Israel Institute of Technology, Haifa, Israel
Meital Suissa, MSc, Technion-Israel Institute of Technology, Haifa, Israel
Clari Valansi, MSc, Technion-Israel Institute of Technology, Haifa, Israel
Wei Wang, MS, David Geffen School of Medicine at UCLA, Los Angeles, CA
Alan J. Waring, PhD, David Geffen School of Medicine at UCLA, Los Angeles, CA
Yongming Xie, PhD, University of California Los Angeles, Los Angeles, CA
For further information, contact lchern@helix.nih.gov.