HIV-1 uncoating by release of viral cDNA from capsid-like structures in the nucleus of infected cells

Abstract

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.

The Role of Capsid in HIV-1 Nuclear Entry

“…Li et al very recently combined GFP-CA labelling with the GFP fluid phase marker and, in contrast to a previous report [ 18 ], found that cores retained integrity until just before integration, suggesting that intact cores were transported through the NPC [ 58 ]. This is supported by recent CLEM experiments that visualised CA assemblies around NPCs, and what looked like intact cores within the nucleus [ 55 ]. These cores were shown to contain viral cDNA as labelled using the ANCHOR system [ 56 ], leading the authors to propose that uncoating happens inside the nucleus by localised physical disruption instead of by cooperative disassembly [ 55 ].…”

“…This is supported by recent CLEM experiments that visualised CA assemblies around NPCs, and what looked like intact cores within the nucleus [ 55 ]. These cores were shown to contain viral cDNA as labelled using the ANCHOR system [ 56 ], leading the authors to propose that uncoating happens inside the nucleus by localised physical disruption instead of by cooperative disassembly [ 55 ]. It is worth noting, however, that in this case, the ANCH sequence-containing viral cDNA must be accessible to the OR-GFP protein, suggesting a partial opening of the CA lattice.…”

“…The limitation of this study is that most of the data were obtained with a CPSF6-binding deficient CA that may not uncoat in an identical manner to wild type virus. In a more recent publication from the same lab, the authors used WT HIV-1 and detected conical and elongated structures that appeared to be open viral cores inside the nucleus of HeLa derived cells [ 55 ].…”

“…Various groups have now reported the presence of CA in the nucleus by fixed immunofluorescence microscopy [ 32 , 43 , 48 , 49 , 55 , 56 , 59 , 60 , 61 ]. Muller et al have recently shown efficient detection of nuclear CA signals in HeLa-derived cells is improved by fixing with methanol or by displacing CPSF6 from subviral complexes using PF74 treatment after nuclear entry has occurred [ 55 ]. In addition, treatment with protease after fixation and permeabilisation, was shown to enhance nuclear CA signal in HeLa-T4 cells [ 49 ].…”

“…This also suggested that reverse transcription may occur inside intact, or nearly intact, cores and that synthesis of double-stranded viral cDNA induced physical changes to the lattice. More recent cell-based studies have confirmed that reverse transcription can occur inside an intact or stabilised core [ 30 , 55 , 57 , 58 , 63 ]. Time-of-addition studies in infected cells showed that sensitivity to RT inhibitors was lost before sensitivity to the CA lattice-binding small molecule, PF74, suggesting that assembled capsids composed of CA hexamers are retained until after completion of reverse transcription [ 30 , 31 , 32 ].…”