FIG 6.

Multiple CA proteins leading retrotranscribed DNA detected by CLEM. (A) HeLa P4R5 cells transduced with OR-GFP were infected with HIV-1 ANCH3 and processed for correlative light and electron microscopy (CLEM). To achieve a precision correlation, fluorescent beads of 200 nm were added, visible also by EM. CLEM results on HeLa P4R5 OR-GFP cells at 6 h postinfection show the GFP signal (green), revealing the location of HIV-1 DNA. The signal of OR-GFP was amplified by using an antibody against GFP. HIV-1 CA was detected by the primary antibody anti-CA and a secondary gold-conjugated antibody (10-nm gold particles). The yellow signals correspond to the beads emitting in the green and in red channels. On the top right, a magnified view of the boxed area is shown. The green signal indicates the location of viral DNA, associated with the gold-labeled CA (dark dots). (B) Another biological replicate of CLEM shows multimers of CA proteins (dark dots) shrouding the vDNA (green signal) during nuclear translocation (left). The panel on the right shows an example of the precision correlation process applied. The precision of the correlation between TEM and fluorescence images was estimated with the ecCLEM plug-in in the Icy software environment. The calibration bar represents the precision achieved (in nanometers) by the different areas of the cells. The dashed circle shows the area enlarged in the boxed area of the panel on the left.