Journal of Biological Chemistry
Volume 295, Issue 45, 6 November 2020, Pages 15183-15195
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HIV-cell membrane fusion intermediates are restricted by Serincs as revealed by cryo-electron and TIRF microscopy

https://doi.org/10.1074/jbc.RA120.014466Get rights and content
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To enter a cell and establish infection, HIV must first fuse its lipid envelope with the host cell plasma membrane. Whereas the process of HIV membrane fusion can be tracked by fluorescence microscopy, the 3D configuration of proteins and lipids at intermediate steps can only be resolved with cryo-electron tomography (cryoET). However, cryoET of whole cells is technically difficult. To overcome this problem, we have adapted giant plasma membrane vesicles (or blebs) from native cell membranes expressing appropriate receptors as targets for fusion with HIV envelope glycoprotein-expressing pseudovirus particles with and without Serinc host restriction factors. The fusion behavior of these particles was probed by TIRF microscopy on bleb-derived supported membranes. Timed snapshots of fusion of the same particles with blebs were examined by cryo-ET. The combination of these methods allowed us to characterize the structures of various intermediates on the fusion pathway and showed that when Serinc3 or Serinc5 (but not Serinc2) were present, later fusion products were more prevalent, suggesting that Serinc3/5 act at multiple steps to prevent progression to full fusion. In addition, the antifungal amphotericin B reversed Serinc restriction, presumably by intercalation into the fusing membranes. Our results provide a highly detailed view of Serinc restriction of HIV-cell membrane fusion and thus extend current structural and functional information on Serinc as a lipid-binding protein.

HIV
membrane fusion
virus entry
host-pathogen interaction
cryo-electron microscopy
single-particle tracking
human immunodeficiency virus (HIV)

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This article contains supporting information.

Author contributions—A. E. W., O. P., J. M. W., B. K. G.-P., and L. K. T. conceptualization; A. E. W. and B. K. G.-P. data curation; A. E. W. formal analysis; A. E. W. and B. K. G.-P. investigation; A. E. W. and B. K. G.-P. visualization; A. E. W. writing-original draft; V. K. resources; V. K. software; V. K. validation; V. K., O. P., and B. K. G.-P. methodology; O. P., J. M. W., B. K. G.-P., and L. K. T. supervision; J. M. W., B. K. G.-P., and L. K. T. writing-review and editing; L. K. T. funding acquisition.

Funding and additional information—This work was supported by National Institutes of Health Grants R01 AI030557 (to L. K. T.), F30 HD101348 (to A. E. W.), and P50 AI150464 (to B. K. G.-P.). The University of Virginia Molecular Electron Microscopy Core is supported in part by NIH grant U24 GM116790. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Abbreviations—The abbreviations used are:

    cryoET

    cryo-electron tomography

    GPMV

    giant plasma membrane vesicle

    TIRF

    total internal reflection fluorescence

    SPPM

    supported planar plasma membrane

    ROI

    region of interest

    DMPE

    dimyristoylphosphatidylethanolamine

    IP6

    inositol hexaphosphate.