Membrane Rearrangements in Cell Fusion and Syncytin 1–Mediated Gene Transfer
- Leonid V. Chernomordik, PhD, Head, Section on Membrane Biology
- Eugenia Leikina, DVM, Senior Research Assistant
- Kamram Melikov, PhD, Staff Scientist
- Elena Zaitseva, PhD, Staff Scientist
- Jarred Whitlock, PhD, Intramural Research Training Award Fellow
- Gracia Luoma-Overstreet, BS, Postbaccalaureate Fellow
Diverse biological processes in which enveloped viruses infect cells and cells from all kingdoms of life secrete, internalize, traffic and sort integral proteins, sculpt their membranes, and bring together parent genomes in sexual reproduction share a common stage: fusion of two membranes into one. Biological membrane remodeling is tightly controlled by protein machinery but is also dependent on the lipid composition of the membranes. Whereas each kind of protein has its own individual personality, membrane lipid bilayers have rather general properties manifested by their resistance to disruption and bending and by their charge. Our long-term goal is to understand how proteins fuse membrane lipid bilayers. We expect a better understanding of important fusion reactions to bring about new ways of controlling them and lead to new strategies for quelling diseases involving cell invasion by enveloped viruses and defects in intracellular trafficking or intercellular fusion. Our general strategy is to combine in-depth analysis of the best-characterized fusion reactions with comparative analysis of diverse, less explored fusion reactions, which can reveal new kinds of fusion proteins and clarify the generality of emerging mechanistic insights. In our recent studies, we explored the mechanisms of horizontal gene transfer from human cells transduced with retroviral vector to non-transduced cells.
Syncytin 1–dependent horizontal transfer of marker genes from retrovirally transduced cells
Retroviral transduction is routinely used to generate cell lines expressing exogenous non-viral genes. Retroviral vectors and cells containing retroviral vectors are considered for clinical applications. It is critically important to minimize the risk of the production of a replication-competent retrovirus (RCR) that may deliver the introduced gene or other genes from the transduced cell to non-transduced cells. To satisfy the latter requirement, the gene transfer plasmid lacks the genes required for γ-retroviral packaging and transduction. During production of a retroviral vector, such genes are provided by other plasmids or are stably expressed in the packaging cell line. Nevertheless, RCRs represent an important safety concern in the development of retroviral gene therapy.
In our recent study, we reported that human cells, transduced to stably express GFP, transfer the GFP gene to non-transduced cells. While this transfer was observed for many different types of donor (retrovirally transduced) cells to several different types of acceptor (non-transduced), the efficiency of the transfer depended on both donor cells and acceptor cells. GFP gene transfer was also observed when co-culture of the cells was replaced by application of the conditioned medium from the transduced cells.
Apparently, all mammalian cells release EVs. Given that, for retrovirus-releasing cells, the physical properties and sizes of many extracellular vesicles (EVs) strongly overlap with those of retroviruses, conventional EV preparations contain both EVs and retroviral particles and will be referred to below as extracellular membrane vesicles (EMV). Non-transduced PC3 cells incubated with EMVs from the cells retrovirally transduced to express GFP developed expression of GFP, with the fraction of the GFP–expressing cells similar to the fractions observed after co-culturing the non-transduced cells with the conditioned medium from the GFP–transduced cells.
We found that EMVs involved in the gene transfer carry endogenous the retroviral envelope protein Syncytin 1 and that the transfer depends on the fusogenic activity of Syncytin 1. Cell types expressing more Syncytin 1 are more efficient as donor cells, and suppression of Syncytin 1 expression in donor cells inhibits the transfer. A Syncytin 1–derived synthetic peptide known to block fusogenic restructuring of Syncytin 1, but not the control scrambled peptide, suppresses the GFP transfer by EMVs collected from the donor cells. The dependence of the gene transfer on the fusogenic activity of Syncytin 1 was further confirmed by inhibition of the transfer by blocking ASCT2 receptor of Syncytin 1 with an ASCT2–binding reagent ASCT2.
Overexpression of Syncytin 1 in the cells transduced with an envelope-defective retroviral sequence has been reported to result in assembly of pseudo-typed viruses with the ability to fuse with cells and to spread viral gene sequences to new target cells. Our findings indicate that even endogenous levels of expression of Syncytin 1 in cells can be sufficient for this protein to be acquired by EMVs and to effect Syncytin 1–mediated fusion of these vesicles. The levels of the expression of both Syncytin 1 and ASCT2 vary widely between different human cells and under different conditions. For instance, expression of Syncytin 1 and ASCT2 is boosted by the cytokines interleukins 4 and 13 and by tumor necrosis factor alpha. Several types of cancer, including prostate adenocarcinoma and endometrial carcinoma, have elevated levels of Syncytin 1 expression, with further upregulation of Syncytin 1 associated with cancer progression. Syncytin 1 expression is also boosted by some viral infections and linked to neurological diseases. Such data suggest that some retrovirally transduced cells can generate EMVs with Syncytin 1–dependent infectivity only under specific conditions, conditions that boost Syncytin 1 expression in these cells or ASCT2 expression in the surrounding cells.
In the context of clinical applications, where generation of RCRs is expected to increase the risk of treatment-related malignancy, emergence of RCRs is a very well-recognized concern. Both vector-producing cells and the transduced cells developed for gene therapy studies are routinely tested for the presence of RCR. Our findings indicate that stable cell lines generated by retroviral vectors in in vitro studies, where the transduction protocols are optimized to achieve the highest efficiency of the transduction by utilizing single packaging vector encoding gag, pol, and env, must also be tested for RCRs. Repetitive integration events that can accompany many infections by RCR can lead to important changes in protein expression profile and cell properties. Undetected and unexpected RCR production in laboratory cell lines presents an additional safety concern, especially given the fact that, in human cell line HERV, env proteins can change the tropism of the produced viral particles.
Lipid mixing assay for myoblast fusion and other slow cell-cell fusion processes
Lipid mixing (redistribution of lipid probes between fusing membranes) has been widely used to study early stages of relatively fast viral and intracellular fusion processes that take seconds to minutes. Lipid mixing assays are especially important for the identification of hemifusion intermediates, operationally defined as lipid mixing without content mixing. Given the unsynchronized character and the slow rate of the differentiation processes that prime the cells for cell-cell fusion processes in myogenesis, osteoclastogenesis and placentogenesis, such fusions take days. Application of lipid-mixing assays to detect early fusion intermediates in these very slow fusion processes must consider the continuous turnover of plasma membrane components and potential fusion-unrelated exchange of the lipid probes between the membranes. We applied a lipid mixing assay in our work on myoblast fusion stage in development and regeneration of skeletal muscle cells. Our approach utilizes a conventional in vitro model of myogenic differentiation and fusion based on murine C2C12 cells. When we observe the appearance of first multinucleated cells, we lift the cells and label them with either the fluorescent lipid DiI as a membrane probe or CellTracker™ Green as a content probe. Redistribution of the probes between the cells is scored by fluorescence microscopy. Hemifused cells are identified as mononucleated cells labeled with both content and membrane probes. The interpretation must be supported by a system of negative controls with fusion-incompetent cells to account for and minimize contributions of fusion-unrelated exchange of the lipid probes. With minor modifications the approach has been used to investigate fusion of primary murine myoblasts and osteoclast precursors, as well as fusion mediated by a gamete fusogen HAP2, and likely can be adopted for other slow cell-cell fusion processes.
Additional Funding
- NICHD Director’s Awards, 2020, 2021
- Office of Aids Research Award, 2019, 2020, 2021
- NICHD’s Strategic Plan 2020
Publications
- Uygur B, Melikov K, Arakelyan A, Margolis LB, Chernomordik LV. Syncytin 1 dependent horizontal transfer of marker genes from retrovirally transduced cells. Sci Rep 2019;9:17637.
- Leikina E, Melikov K, Rabinovich AG, Millay D, Chernomordik LV. Lipid mixing assay for murine myoblast fusion and other slow cell-cell fusion processes. Bio Protoc 2020;10:3544.
Collaborators
- Anush Arakelyan, PhD, Section on Intercellular Interactions, NICHD, Bethesda, MD
- Michael M. Kozlov, PhD, Dhabil, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
- Leonid Margolis, PhD, Section on Intercellular Interactions, NICHD, Bethesda, MD
- Douglas Millay, PhD, Cincinnati Children's Hospital Medical Center, Cincinnati, OH
- Benjamin Podbilewicz, PhD, Technion-Israel Institute of Technology, Haifa, Israel
Contact
For more information, email chernoml@mail.nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/chernomordik.
