TY - JOUR TI - HIV-1 uncoating by release of viral cDNA from capsid-like structures in the nucleus of infected cells AU - Müller, Thorsten G AU - Zila, Vojtech AU - Peters, Kyra AU - Schifferdecker, Sandra AU - Stanic, Mia AU - Lucic, Bojana AU - Laketa, Vibor AU - Lusic, Marina AU - Müller, Barbara AU - Kräusslich, Hans-Georg A2 - Sundquist, Wesley I A2 - Sawyer, Sara L A2 - Campbell, Edward M VL - 10 PY - 2021 DA - 2021/04/27 SP - e64776 C1 - eLife 2021;10:e64776 DO - 10.7554/eLife.64776 UR - https://doi.org/10.7554/eLife.64776 AB - HIV-1 replication commences inside the cone-shaped viral capsid, but timing, localization, and mechanism of uncoating are under debate. We adapted a strategy to visualize individual reverse-transcribed HIV-1 cDNA molecules and their association with viral and cellular proteins using fluorescence and correlative-light-and-electron-microscopy (CLEM). We specifically detected HIV-1 cDNA inside nuclei, but not in the cytoplasm. Nuclear cDNA initially co-localized with a fluorescent integrase fusion (IN-FP) and the viral CA (capsid) protein, but cDNA-punctae separated from IN-FP/CA over time. This phenotype was conserved in primary HIV-1 target cells, with nuclear HIV-1 complexes exhibiting strong CA-signals in all cell types. CLEM revealed cone-shaped HIV-1 capsid-like structures and apparently broken capsid-remnants at the position of IN-FP signals and elongated chromatin-like structures in the position of viral cDNA punctae lacking IN-FP. Our data argue for nuclear uncoating by physical disruption rather than cooperative disassembly of the CA-lattice, followed by physical separation from the pre-integration complex. KW - HIV-1 KW - uncoating KW - reverse transcription KW - DNA labeling KW - live cell imaging KW - CLEM JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -