Background: We have investigated the structure of HIV-1 intracellular reverse transcription complexes (RTCs) by confocal and transmission electron microscopy (TEM).

Methods: Cytoplasmic extracts were prepared 3, 4, and 16 hrs post-acute infection by dounce homogenisation in hypotonic buffer without detergents. RTCs were purified by velocity sedimentation followed by density fractionation in linear sucrose gradients and dialysis in a large pore cellulose membrane. Uninfected cells were processed in parallel and used as negative control. Purified RTCs were competent to reverse transcribe in vitro. Double labelling of nucleic acids and viral proteins allowed the specific identification of RTCs by confocal microscopy.

Results: RTCs have a sedimentation velocity of 350S and a density of 1.34 g/ml. Electron and confocal microscopy analyses revealed that RTCs are large nucleoprotein structures approximately 250–300 nm long and 100–150 nm widths mainly composed of semi-condensed filaments approximately 6 nm thick. Treatment of RTCs with proteinase K indicated that the 6 nm filaments contain the nucleic acids, which are associated to small proteins. Nucleic acids in the complex are partially protected from nuclease digestion, but they are not organised into nucleosomes. Vpr and integrase are bound to the 6 nm filament as early as 3 hrs post-infection. The amount and distribution of Vpr, integrase, and NC proteins in the RTC was examined by immuno-EM and the results suggest that cellular proteins are also associated to the viral nucleic acids.

Conclusions: Our data indicate that a partially condensed nucleoprotein complex that lacks a core-derived shell mediates reverse transcription of the HIV-1 genome. Integrase, Vpr, and other small proteins bind to the viral nucleic acids and may play a role in reverse transcription and in preventing RTC degradation.