Rapid formation of human immunodeficiency virus-like particles
| Autor: | Pavel Novák, Andrew Shevchuk, Joanna Bednarska, Peter A. Summers, Annegret Pelchen-Matthews, Mark Marsh, Jemima J. Burden, Marina K. Kuimova, Yuri E. Korchev |
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| Přispěvatelé: | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
assembly
MECHANISM Fluorescence-lifetime imaging microscopy SURFACE PROTEINS viruses MEMBRANES Microbiology gag Gene Products Human Immunodeficiency Virus Virus Green fluorescent protein law.invention Cell Line Cell membrane Viral envelope Confocal microscopy law medicine Humans LIVING CELLS Virus Release GAG Budding Multidisciplinary Science & Technology Chemistry Virus Assembly Cell Membrane Virion HIV ASSOCIATION Biological Sciences Multidisciplinary Sciences SICM medicine.anatomical_structure Biophysics HIV-1 Science & Technology - Other Topics VISUALIZATION VISCOSITY HIV-1 RELEASE |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America |
| Popis: | Significance Currently, our knowledge of individual virus particle assembly kinetics is based solely on studies of the dynamics of fluorescent puncta corresponding to the viral structural protein called Gag labeled with fluorescent proteins (e.g., green fluorescent protein [GFP]) observed by fluorescence microscopy in membranes of cells. However, GFP tagging affects virus particle assembly and release. We directly measured topological changes during HIV-like particle assembly and found that they can reach full size in 20 s and release in 0.5 to 3 min. Compared to previous estimates of ∼8-min assembly time and 30- to 60-min release time, this is more than 10 times faster. In our opinion this is a highly important discovery that challenges current views on virus replication mechanisms. Understanding the molecular mechanisms involved in the assembly of viruses is essential for discerning how viruses transmit from cell to cell and host to host. Although molecular aspects of assembly have been studied for many viruses, we still have little information about these events in real time. Enveloped viruses such as HIV that assemble at, and bud from, the plasma membrane have been studied in some detail using live cell fluorescence imaging techniques; however, these approaches provide little information about the real-time morphological changes that take place as viral components come together to form individual virus particles. Here we used correlative scanning ion conductance microscopy and fluorescence confocal microscopy to measure the topological changes, together with the recruitment of fluorescently labeled viral proteins such as Gag and Vpr, during the assembly and release of individual HIV virus-like particles (VLPs) from the top, nonadherent surfaces of living cells. We show that 1) labeling of viral proteins with green fluorescent protein affects particle formation, 2) the kinetics of particle assembly on different plasma membrane domains can vary, possibly as a consequence of differences in membrane biophysical properties, and 3) VLPs budding from the top, unimpeded surface of cells can reach full size in 20 s and disappear from the budding site in 0.5 to 3 min from the moment curvature is initially detected, significantly faster than has been previously reported. |
| Databáze: | OpenAIRE |
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