Journal: Scientific reports

Article Title: HIV-1 enhances mTORC1 activity and repositions lysosomes to the periphery by co-opting Rag GTPases.

doi: 10.1038/s41598-017-05410-0

Figure Lengend Snippet: Figure 2. mTOR activity is required for optimal viral-protein synthesis. (a) Schematic of experimental protocol. (b) HeLa cells were transfected with pNL4-3, left untreated or treated with 250 nM Torin1 in medium containing AHA. At the indicated times, cell lysates were collected and Click-it chemistry was performed followed by immunoprecipitation with rabbit anti-p24 antibody. Membranes were probed with the indicated antibodies. (c) Densitometry quantification of de novo synthesized, biotinylated Gag from panel b was determined by ImageJ analysis. For each time point, the values presented in the graph are normalized against the total amount of Gag in the cell lysate (refer to Supplementary Fig. 1 for total protein synthesis). Full-length blots are shown in Supplementary Fig. 5. (d) HeLa cells were transfected with either pcDNA3.1 or pNL4-3 and infected with L. major (lmj) WT or GP63 KO. Cell lysates were subjected to SDS-PAGE, immunoblotted and probed with the indicated antibodies against host (left panels) and viral (right panels) antigens.

Article Snippet: The labeled material was precleared with normal rabbit serum and 25 μL of a 50:50 slurry of protein G-Sepharose (Thermo Scientific), incubated with rabbit anti-p24 (Fitzgerald) for 16 h at 4 °C and with 30 μL of a 50:50 slurry of protein G-Sepharose for 3 h at room temperature.

Techniques: Activity Assay, Transfection, Immunoprecipitation, Synthesized, Infection, SDS Page

Journal: Scientific reports

Article Title: HIV-1 enhances mTORC1 activity and repositions lysosomes to the periphery by co-opting Rag GTPases.

doi: 10.1038/s41598-017-05410-0

Figure Lengend Snippet: Figure 5. RagA and RagB are necessary for HIV-1 to control LEL trafficking and for the viral budding and release. (a) HeLa cells, treated with non-silencing (NS) siRNA or siRNAs targeting RagA and RagB mRNAs, were transfected with empty vector control, pCDNA3.1 or HIV-1 using pNL4-3/GagmRFP, and then mock treated or treated with Ars. Cells were stained for mTOR (Cell Signaling 7C10) and LAMP-1. Light blue arrowheads identify HIV-1-expressing cells, while HIV-1-negative cells are indicated with green arrowheads. A dashed line contours individual cells. Scale bars are 10 μm. (b) The graphs represent the fluorescence intensities of mTOR (green) and LAMP-1 (blue) across the dashed lines from panel a. (c) The percentage of mock or HIV-1-transfected cells showing perinuclear clustering of LEL from panel a. The results are presented as the mean ± S.D. from three different experiments. P value is indicated by *(p < 0.05); n.s.: no signifcant difference between the means. (d) HeLa cells were co-transfected with the HIV-1 pNL4-3 and either non- sense (NS) siRNA or siRNA against RagA and RagB. HIV-1 particles were collected from the supernatant after filtration and ultracentrifugation. Cell lysates and virus particles were separated by SDS-PAGE, transferred to nitrocellulose and probed with the indicated antibodies. siRNA-mediated fold changes in RagA, RagB and virus production are indicated below insets in panel d in this representative experiment. Full-length blots are shown in Supplementary Fig. 5. (e) HeLa cells were co-transfected with the HIV-1 pNL4-3 and either non-sense (NS) siRNA or siRNA against RagA and RagB. Cells were fixed 24 h after transfection and stained for p24. (f) The percentage of HIV-1 and siRagA/B transfected cells showing peripheral accumulation of Gag from e. The results are presented as the mean ± S.D. from three different experiments. (g) HeLa cells were transfected as in e and subjected to transmission electron microscopy.

Article Snippet: The labeled material was precleared with normal rabbit serum and 25 μL of a 50:50 slurry of protein G-Sepharose (Thermo Scientific), incubated with rabbit anti-p24 (Fitzgerald) for 16 h at 4 °C and with 30 μL of a 50:50 slurry of protein G-Sepharose for 3 h at room temperature.

Techniques: Control, Transfection, Plasmid Preparation, Staining, Expressing, Fluorescence, Filtration, Virus, SDS Page, Transmission Assay, Electron Microscopy

Journal: Scientific reports

Article Title: HIV-1 enhances mTORC1 activity and repositions lysosomes to the periphery by co-opting Rag GTPases.

doi: 10.1038/s41598-017-05410-0

Figure Lengend Snippet: Figure 6. RagA and RagB interact with Gag and Vif. (a) Lysates from control pcDNA3.1- or pNL4-3- transfected HeLa cells were subjected to immunoprecipitation with RagA antibodies. Samples were subjected to SDS-PAGE followed by transfer to nitrocellulose membranes and probed with the indicated antibodies. Full- length blots are shown in Supplementary Fig. 5. (b) Lysate from pcDNA3.1-, pNL4-3-, pNLXX−, pNLXX+ pSVGag− and pNLVif–transfected HeLa cells were subjected to immunoprecipitation with RagA and RagB antibodies or isotype rabbit IgG control. The blots were probed with anti-p24, anti-Vif, anti-RagA and anti- RagB antibodies. Full-length blots are shown in Supplementary Fig. 5. (c) HeLa cells were transfected with either pNLXX or pNLVif- and left untreated (Unt) or stressed with 500 µM Ars for 1 h. HIV-1-expressing cells were identified using FISH to localize vRNA and stained using antibodies against mTOR (Cell Signaling) and LAMP-1. Light blue arrowheads identify pNLXX or pNLVif- expressing cells, while pNLXX or pNLVif- negative cells in the same field are indicated with green arrowheads. A dashed line contours individual cells. Scale bars are 10 μm. (d) The percentage of mock, pNLXX or pNLVif- transfected cells showing perinuclear clustering of mTOR/LAMP-1 from panel c. The results are presented as the mean ± S.D. from three different experiments. P value is indicated by *(p < 0.05).

Article Snippet: The labeled material was precleared with normal rabbit serum and 25 μL of a 50:50 slurry of protein G-Sepharose (Thermo Scientific), incubated with rabbit anti-p24 (Fitzgerald) for 16 h at 4 °C and with 30 μL of a 50:50 slurry of protein G-Sepharose for 3 h at room temperature.

Techniques: Control, Transfection, Immunoprecipitation, SDS Page, Expressing, Staining

Journal: bioRxiv

Article Title: The sequestration of miR-642a-3p by a complex formed by HIV-1 Gag and human Dicer increases AFF4 expression and viral production

doi: 10.1101/2023.05.24.542197

Figure Lengend Snippet: RNAi is functional and the localizations of its contributors remain unchanged in HIV-expressing cells. (A) Silencing of EGFP expression by shGFP and miGFP remains functional in HIV-1 expressing cells. HeLa cells were co- transfected with 2 µg of pcDNA3 or pNL4-3, 1 µg of pEGFP-C1 and 4µg of shRNA-NS, shRNA-EGFP, miRNA-NS or miRNA-EGFP, as indicated. EGFP, HIV-1 Gag and actin expression were observed by immunoblot and EGFP silencing effects in relation to paired nonsense controls were quantified by densitometry analysis. (B) Inhibition of endogenous and transfected GAPDH expression by shGAPDH is functional in HIV- expressing cells. HeLa cells were co-transfected with 2 µg pcDNA3 or pNL4-3, 1 µg GAPDH-ProLabel and 1 µg pSIREN-shRNA-LucIRR or pSIREN-shRNA-GAPDHB. GAPDH, HIV-1 Gag and actin expression were observed by immunoblot and GAPDH silencing effects in relation to paired nonsense controls were quantified by densitometry analysis. (C) Inhibition of EGFP expression by let7 is functional in HIV-1 replicating cells. HeLa-CD4 (MAGI) cells were not infected (lanes 1, 2) or infected with pNL4-3 at one million RT cpms (lanes 3, 4) or 2 million RT cpms (lanes 5, 6) of supernatant. They were co-transfected with pEGFP or pEGFP-clet7 24 h later. EGFP, HIV-1 Gag and actin expression were observed by immunoblot and EGFP silencing effects in relation to paired nonsense controls were quantified by densitometry analysis. (D) The number of large GWBs is not altered in HIV-1 expressing cells. HeLa cells were transfected with 0.2 µg of HIV-1 pNL4-3. Cells were fixed and stained 48 h later with the human GWB marker serum 18033 and an antibody targeting HIV-1 p24. Side-by-side images of p24 positive (+ve) and negative (-ve) cells were identified and images were adjusted to visualize only large 18033 stained GWBs, which were then counted. The bar graph shows the mean number of GWBs from 12 pairs of cells. Statistical analysis was performed using an unpaired Mann-Whitney test (p=0.7) between +ve or –ve cells. (E-H) The localization of miRNA biogenesis enzymes and RNAi proteins in the cytoplasm is not altered in HIV-1 expressing cells . HeLa cells were transfected, fixed and stained as in (D) with the addition of antibodies against DDX6 (E), Ago2 (F), TRBP (G) or Dicer (H). The size scale is 20 µm and is shown in each picture on the bottom left. Digitally zoomed images of the merged channels are shown on the far right, with arrows highlighting colocalization between the GWB marker 18033 and DDX6 (E) or Ago2 (F), shown as cyan and purple dots, respectively.

Article Snippet: Cells were incubated with the primary antibodies for 1 h at 37 ℃ using mouse monoclonal anti-p24 (183-H12-5C) ( ) at a dilution of 1/400 or 1/1000, rabbit anti-p24 (from Dr. L. Kleiman) at a 1/1000 dilution, rabbit anti- Rck/p54/DDX6 (Bethyl labs) at a 1/1000 dilution, mouse anti-Ago2 (Wako 4G8) at a 1/100 dilution, rabbit anti-TRBP673 ( ) at a 1/250 dilution, rabbit anti-Dicer 349 ( ) at a 1/250 dilution, mouse monoclonal anti-Dicer 13D6 antibody (Abcam) at a 1/500 dilution and human autoimmune serum 18033 ( ) at a 1/5000 dilution.

Techniques: Functional Assay, Expressing, Transfection, shRNA, Western Blot, Inhibition, Infection, Staining, Marker, MANN-WHITNEY

Journal: bioRxiv

Article Title: The sequestration of miR-642a-3p by a complex formed by HIV-1 Gag and human Dicer increases AFF4 expression and viral production

doi: 10.1101/2023.05.24.542197

Figure Lengend Snippet: GWB counts in side-by-side images of p24 +ve and –ve cells. HeLa cells were transfected with 0.2 μg of HIV-1 pNL4-3. 48 h later cells were fixed and stained with the human GWB marker serum 18033 (red), an antibody targeting HIV-1 p24 (blue) and an antibody targeting TRBP, Ago2 or Dicer (green). The level of red was adjusted with Adobe Photoshop so that only large GWBs (2-14 per cell) were visible. GWBs were then counted in side-by-side images of p24 –ve/+ve cells and recorded below the images.

Article Snippet: Cells were incubated with the primary antibodies for 1 h at 37 ℃ using mouse monoclonal anti-p24 (183-H12-5C) ( ) at a dilution of 1/400 or 1/1000, rabbit anti-p24 (from Dr. L. Kleiman) at a 1/1000 dilution, rabbit anti- Rck/p54/DDX6 (Bethyl labs) at a 1/1000 dilution, mouse anti-Ago2 (Wako 4G8) at a 1/100 dilution, rabbit anti-TRBP673 ( ) at a 1/250 dilution, rabbit anti-Dicer 349 ( ) at a 1/250 dilution, mouse monoclonal anti-Dicer 13D6 antibody (Abcam) at a 1/500 dilution and human autoimmune serum 18033 ( ) at a 1/5000 dilution.

Techniques: Transfection, Staining, Marker

Journal: bioRxiv

Article Title: The sequestration of miR-642a-3p by a complex formed by HIV-1 Gag and human Dicer increases AFF4 expression and viral production

doi: 10.1101/2023.05.24.542197

Figure Lengend Snippet: (Top) In the absence of HIV-1, miR-642a-3p is loaded on Dicer-TRBP, which recruits Ago2 to form the RNA-induced silencing complex (RISC). miR-642a-3p targets and silences AFF4 mRNA, resulting in a poor assembly of the super elongation complex (SEC) and paused RNA polymerase (Pol) II. (Bottom) In the presence of HIV-1, Gag binds to Dicer, which induces the sequestration of miR-642a- 3p and prevents its loading on the RISC. AFF4 mRNA is translated, which results in the formation of an active SEC including AFF4 and a processive RNA Pol II.

Article Snippet: Cells were incubated with the primary antibodies for 1 h at 37 ℃ using mouse monoclonal anti-p24 (183-H12-5C) ( ) at a dilution of 1/400 or 1/1000, rabbit anti-p24 (from Dr. L. Kleiman) at a 1/1000 dilution, rabbit anti- Rck/p54/DDX6 (Bethyl labs) at a 1/1000 dilution, mouse anti-Ago2 (Wako 4G8) at a 1/100 dilution, rabbit anti-TRBP673 ( ) at a 1/250 dilution, rabbit anti-Dicer 349 ( ) at a 1/250 dilution, mouse monoclonal anti-Dicer 13D6 antibody (Abcam) at a 1/500 dilution and human autoimmune serum 18033 ( ) at a 1/5000 dilution.

Techniques: