tRNAs Promote Nuclear Import of HIV-1 Intracellular Reverse Transcription Complexes
Figure 6
Structural Requirements of tRNAs for RTC Nuclear Import Activity
(A) Quantification of RTC nuclear import in the presence of G2 RNA, modified G2 RNA, and modified G2 RNA with 3′ CCA end. Quantification was performed as in (D) and bars represent the mean log fluorescence value of the nuclei ± standard deviation of five independent experiments. Correct generation of the tRNALys1,2-size G2 RNA and addition of the CCA tail to this RNA was monitored by long 15% denaturing PAGE followed by SYBR Gold staining (bottom panel).
(B) The presence of the correct 3′ CCA tail in the tRNA Lys1,2 + CCA molecule was monitored by splint labelling. The tRNA Lys1,2 + CCA RNA was purified by denaturing PAGE as shown in (A), bottom. Following splint labelling with [α-32P]dATP, the G2 + CCA RNA was re-analyzed on a 15% denaturing PAGE and visualized by PhosphoImager (see Protocol S1). Labelling was performed with antisense primer −CCA (lane 1), and with antisense primer +CCA (lane 2).
(C) Nuclear import of YOYO-1–labelled RTC in permeabilized HeLa cells in the presence of 1× energy-regenerating system and mutant RNAs generated by in vitro T7 transcription (140 nM each) (see Tables S1 and S2) or buffer (ctr–).
G2, original RNA; G2nAS, mutant with shortened acceptor stem; G3, original RNA; G3 C56U, point mutation disrupting D- and T-loops pairing; G3 C62G + A64C, double point mutation disrupting T-stem secondary structure; G3 AnC-T, deletion mutant lacking the D-arm; G3D, deletion mutant lacking anticodon loop and T-arm; G3T, deletion mutant lacking D- and anticodon arms.
(D) Quantification of RTC nuclear import with the same RNA mutants as shown in (C). Images were acquired by confocal microscopy and analyzed using the MetaMorph software version 4.5r4 (Universal Imaging Corp) and the total fluorescence in the nucleus divided by the number of cells per field. At least 150 cells were counted per experiment. Bars represent the percentage of total fluorescence per nucleus relative to control (G2 and G3 = 100) ± standard deviation of three independent experiments.