(A) Western blot showing depletion of both imp7 and Ran in the supernatant (60S) of the 60% AS precipitation step and depletion of imp7 but not Ran from the 60% AS precipitation pellet (60P) after low-substitution Phenyl-Sepharose chromatography (d60P).
(B) Nuclear import in permeabilized HeLa cells in the presence of labelled RTCs, 1× energy-regenerating system and buffer (panel ctr−), 1μM imp7 + 1× Ran mix (panel imp7), 0.5 mg/ml of the pellet (panel 60P) or supernatant (panel 60S) fractions from the 60% AS precipitation step, or 60P after low-substitution Phenyl-Sepharose chromatography (panel d60P). Images were acquired by confocal microscopy using the same settings. Scale bar indicates 25 μm.

(A) Equal amounts of nucleic acids purified from the active Phenyl-Sepharose fraction (60S NA) were subjected to nuclease S7 (NS7) or DNAse-free RNAse (RNAse) treatment and analyzed by 15% denaturing PAGE followed by silver staining. Total HeLa RNA (totRNA) was used as control for nuclease and RNAse treatments.
ST1, oligonucleotide size markers (range, 8–32 nucleotides); ST2, size markers pBP322DNA-MspI.
(B) 60S NA loses its ability stimulate RTC nuclear import after nuclease or RNAse treatment. Nuclear import of YOYO-1 labelled RTCs in permeabilized primary human macrophages in the presence of 1× energy-regenerating system and 60S (0.5 mg/ml), 60S NA (1 μg), 60S NA digested with NS7, 60S NA digested with RNAse (1-μg starting material), 21mer siRNA (1 μg), total HeLa RNA (totRNA, 1μg), or buffer (ctr –).
(C) 60S NA fraction can be specifically 3′-end radiolabelled by T4 RNA ligase. Following 3′-end labelling with 5′-[³²P]pCp, samples were analyzed by 15% denaturing PAGE and visualized by Storm 860 PhosphoImager. Total HeLa RNA (totRNA) was used as a control for T4 RNA ligase reaction. ST1 and ST2 are 5′-end radiolabelled oligonucleotide size markers as in (A).

(A) Nuclear import of YOYO-1–labelled RTCs in permeabilized HeLa cells in the presence of 1× energy-regenerating system and Fr2 (100 ng), the indicated small RNAs generated by in vitro T7 transcription (see Table 1, Protocol S1, and Table S2) (140 nM each, corresponding to ~100 ng), human tRNA^Lys1,2 (100 ng), bovine tRNAs (100 ng), or buffer (ctr–). Nuclear import of the G3 RNA molecule in the absence of YOYO-1–labelled RTCs (G3 − RTC) was used as an additional negative control.
(B) Quantification of RTC nuclear import as shown in (A). Images acquired by confocal microscopy were analyzed by MetaMorph software version 4.5r4 (Universal Imaging Corp) and the total fluorescence of the nuclei divided by the number of cells per field (see also Figure S2). At least 150 cells were counted per experiment. Bars represent the log mean fluorescence per nucleus ± standard deviation of six independent experiments.
(C) Quantification of G2/G3 RNA in HSE NA and 60S NA by Northern blot. RNA was separated by 15% denaturing PAGE, transferred onto a nylon membrane, and probed with an oligonucleotide complementary to G2/G3 RNA. There were 100-ng G3 RNA (lane 1), 10-ng G3 RNA (lane 2), 100-ng negative control A9 RNA (tRNA^Gly) (lane 3), 700-ng HSE NA (lane 4), 350-ng HSE NA (lane 5), 175-ng HSE NA (lane 6), and 200-ng 60S NA (lane 7). Upper panel, hybridized membrane visualized by Phosphoimager, lower panel, SYBR Gold staining of denaturing PAGE. Image Quant (Molecular Dynamics) was used to calculate the intensity of the signals.
(D) Comparative RTC nuclear import activity of HSE NA (100 ng), 60S NA (100 ng), G3 RNA (100 ng), G3 RNA (10 ng + 90-ng carrier siRNA), buffer (ctr–), or buffer + 100-ng carrier RNA. Nuclear import assays were performed in permeabilized HeLa cells in the presence of labelled RTCs, 1× energy mix, and the indicated RNAs. Bars represent the fold nuclear import increase above background (RTC + energy mix) ± standard deviation of two independent experiments.

(A) Nuclear import assay in permeabilized HeLa cells in the presence or absence of 1× energy-regenerating system and YOYO-1–labelled RNA molecules and their mutants (see Tables S1 and S2 and Figure 6) (140 nM each, corresponding to 100 ng), human tRNA^Lys1,2 (100 ng), and bovine tRNAs (100 ng). Nuclear import assay of the G3 D mutant RNA was performed in the presence of 100 ng inactive carrier RNA (21mer RNA).
(B) Nuclear import assay in the presence of 140 nM YOYO-1–labelled G3 RNA performed at different temperatures with or without an energy-regenerating system.
(C) Nuclear import assay with labelled RTCs and buffer (RTC) or 140 nM G3 RNA (RTC + G3) performed at different temperatures in the presence of an energy-regenerating system.

(A) Viral RNA (1.5 μg) was extracted from purified HIV-1 (lane 1) or MHIV-1 gag mutant (lane 2) and separated onto a long (50 cm) 15 % denaturing PAGE followed by SYBR Gold staining. There was a 10-bp DNA ladder (lane St), 25-ng tRNA^Lys1,2 size G2 RNA + C tail (see Table S2) (lane 3), 25-ng tRNA^Lys1,2 size G2 RNA + CC tail (lane 4), and 25-ng tRNA^Lys1,2 size G2 RNA + CCA tail (lane 5). Asterisks indicate the three small RNA bands found in HIV-1; arrows indicate viral-specific small RNA molecule common to both viruses.
(B) Single-cycle infection assays in cell cycle–arrested cells. Cells were treated with aphidicolin for 24 h to induce G1/S arrest, infected with the same dose of wild-type HIV-1 or MHIV gag mutant, and analyzed for GFP expression by flow cytometry 24 h after infection. Bars represent the average value ± standard deviation of two experiments.
(C) Nuclear import of YOYO-1–labelled HIV-1 RTCs in permeabilized HeLa cells in the presence of 1× energy-regenerating system and buffer (ctr–) or HIV-1 small RNAs (HIV-1 sRNA) or MHIV-1 gag mutant small RNAs (MLV/HIV-1 sRNA) (30ng + 70-ng carrier siRNA) after elution from the gel. Nuclear import of the eluted small viral RNAs from wild-type HIV-1 and MHIV gag mutant in the absence RTCs was performed as an additional negative control (HIV-1 sRNA and MLV/HIV sRNA, respectively).

(A) Chromatographic profile obtained after hydrophobic interaction and ion exchange chromatography of the original 60S (see Materials and Methods). Individual fractions were analyzed by SDS-PAGE and silver staining (60S, 20-μg protein/lane, and Fractions (Fr) A to F, 10-μg protein/lane). Asterisks indicate the two bands present in FrE.
(B) Nuclear import of YOYO-1–labelled RTCs in permeabilized HeLa cells in the presence of 1× energy-regenerating system, 60S, fractions FrA to FrF (0.5 mg/ml) or buffer (ctr –).
(C) The two bands in FrE are nucleic acids. Equal amounts of FrE were subjected to proteinase K (PK) or nuclease S7 (NS7) treatment (see Materials and Methods) and analyzed by SDS-PAGE or native PAGE followed by silver staining. Total HeLa RNA (totRNA) and RNAse B were used as controls for nuclease or proteinase digestions, respectively.

(A) The 60S NA was separated onto a 15% denaturing gel, and individual bands were cut, eluted from the gel, and re-loaded onto a 15% denaturing gel to confirm correct size fractionation. Bands were labelled by SYBR gold staining and visualized by Storm 860 PhosphoImager. ST1 and ST2, oligonucleotide size markers, as in Figure 3A.
(B) Nuclear import YOYO-1–labelled RTCs in permeabilized HeLa cells in the presence of 1× energy-regenerating system and 60S (0.5 mg/ml), 60S NA (100 ng), the nine main bands eluted from the gel as shown in (A) (140 nM each), or buffer (ctr–).

(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 tRNA^Lys1,2-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.

Total and small RNA were obtained from cells and purified virions, and analyzed onto a long (50 cm) 15% denaturing PAGE followed by SYBR Gold staining. There was a 10-bp DNA ladder (lane St), 2.3-μg total 293T RNA (lane 1), 1.5-μg small 293T RNA (lane 2), purified HIV-1 virion RNA (169 ng and 338 ng) (lanes 3 and 4, respectively), purified RNA from HIV-1 virions not containing the viral genome (472 ng and 236 ng) (lanes 5 and 6, respectively), 2.7-μg total HeLa RNA (lane 7), 2.4-μg small HeLa RNA (lane 8), 100-ng 60S NA fraction from HeLa cells (lane 9), 100-ng Fr2 from HeLa cells (lane 10), 0.9-μg SupT1 small RNA (lane 11), 200-ng 60S NA fraction from SupT1 cells (lane 12), 200-ng Fr2 from SupT1 cells (lane 13), 1.5-μg bovine tRNA (lane 14), 200-ng tRNA^Lys1,2 size G2 RNA + C tail (see Table S2) (lane 15), 200-ng tRNA^Lys1,2 size G2 RNA + CC tail (lane 16), and 200-ng tRNA^Lys1,2 size G2 RNA + CCA tail (lane 17). Asterisks indicate positions of similar size bands in different lanes. Arrows indicate small RNAs specifically enriched in purified virions. The contrast in lanes 3 to 6 in the magnified rectangle has been artificially increased to help in visualizing bands.