The 60S NA Fraction Contains Small RNA Molecules

<div><p>(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.</p> <p>ST1, oligonucleotide size markers (range, 8–32 nucleotides); ST2, size markers pBP322DNA-MspI.</p> <p>(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 –).</p> <p>(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).</p></div

MHIV-1 <i>gag</i> Mutant Does Not Incorporate tRNA Species with RTC Nuclear Import Activity

<div><p>(A) Viral RNA (1.5 μg) was extracted from purified HIV-1 (lane 1) or MHIV-1 <i>gag</i> 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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#pbio-0040332-st002" target="_blank">Table S2</a>) (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.</p> <p>(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 <i>gag</i> mutant, and analyzed for GFP expression by flow cytometry 24 h after infection. Bars represent the average value ± standard deviation of two experiments.</p> <p>(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 <i>gag</i> 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 <i>gag</i> mutant in the absence RTCs was performed as an additional negative control (HIV-1 sRNA and MLV/HIV sRNA, respectively).</p></div

Nuclear Import Activity of Small RNA Molecules Generated In Vitro

<div><p>(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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#pbio-0040332-t001" target="_blank">Table 1</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#pbio-0040332-sd001" target="_blank">Protocol S1</a>, and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#pbio-0040332-st002" target="_blank">Table S2</a>) (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.</p> <p>(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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#pbio-0040332-sg002" target="_blank">Figure S2</a>). At least 150 cells were counted per experiment. Bars represent the log mean fluorescence per nucleus ± standard deviation of six independent experiments.</p> <p>(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.</p> <p>(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.</p></div

Purification and Characterization of the Active Component in the 60S

<div><p>(A) Chromatographic profile obtained after hydrophobic interaction and ion exchange chromatography of the original 60S (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#s4" target="_blank">Materials and Methods</a>). 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.</p> <p>(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 –).</p> <p>(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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#s4" target="_blank">Materials and Methods</a>) 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.</p></div

Size Fractionation of the Small RNA Molecules in the 60S NA

<div><p>(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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#pbio-0040332-g003" target="_blank">Figure 3</a>A.</p> <p>(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–).</p></div

In Vitro Generated tRNAs Accumulate into the Nucleus of Permeabilized HeLa Cells in an Energy- and Temperature-Dependent Way

<div><p>(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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#pbio-0040332-st001" target="_blank">Tables S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#pbio-0040332-st002" target="_blank">S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#pbio-0040332-g006" target="_blank">Figure 6</a>) (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).</p> <p>(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.</p> <p>(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.</p></div

Profile of Small RNA Species Incorporated into HIV-1 Particles

<p>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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#pbio-0040332-st002" target="_blank">Table S2</a>) (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.</p

imp7 and Additional Cytosolic Factors Support RTC Nuclear Import

<div><p>(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).</p> <p>(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.</p></div

Hyperthermia increases infection with single-cycle HIV-1 but not with a GFP lentivector.

<p>A: Infection of Jurkat cells with single-cycle HIV. One representative experiment is shown. Cells were infected with 5, 20, or 100 ng Gag p24 of Δ<i>env</i>(VSV)/mL/10⁶ cells for 2 hours at 37°C or 39.5°C, washed, and grown at 37°C or 39.5°C. Gag levels were assessed by flow cytometry at 24 hours p.i. B: Infection of P4C5 cells with single-cycle HIV. One representative experiment is shown. 8×10⁴ P4C5 cells were plated in 96-well plates. Cells were infected in triplicates with 1 or 5 ng Gag p24 of Δ<i>env</i>(VSV) and grown at 37°C or 39.5°<i>C</i>. Infection was assessed 36 hours p.i. by measuring β-Galactosidase activity (570 nm OD). C: Mean ± SD of 4 independent experiments. P4C5 cells were infected with 1 ng Gag p24 of the indicated HIV strains. Statistical significance was assessed by the Mann-Withney test. p<0.05(*). D: Infection of P4C5 cells at different temperatures. One of two representative experiments is shown. Data are mean ± SD of triplicates. Using the same protocol as in B, cells were infected with 5 ng Gag p24 of NL4-3 and grown at the indicated temperatures. E: Transduction of P4C5 and Jurkat cells with a GFP expressing lentivector (LV-GFP). One of three representative experiments is shown. P4C5 or Jurkat cells were transduced with 1, 10 or 100 ng Gag p24/mL/10⁶ cells of LV-GFP. GFP levels were measured 24 hours later by flow cytometry.</p

Hyperthermia increases viral reactivation in J-Lat 10.6 cells.

<p>A: Experimental outline. J-Lat 10.6 are Jurkat-derived cells carrying a latent, integrated, provirus, encoding <i>gfp</i> instead of <i>nef</i>. Stimulation of J-Lat 10.6 cells with TNFα triggers HIV-1 reactivation and GFP expression. B: Reactivation of J-Lat 10.6 cells with various stimuli. One representative experiment is shown. J-Lat 10.6 cells were treated with either TNFα, supernatants from IL-2 treated or PHA-activated PBMCs, or co-cultivated with IL-2-treated PBMCs for 48 hours at 37°C or 39.5°C. Viral reactivation was assessed by measuring GFP levels by flow cytometry. C: Mean ± SD of 4 independent experiments. Statistical significance was assessed by a paired t test. p<0.05(*).</p