12 research outputs found
Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging
<div><p>During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected <i>in situ</i> as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing unspliced HIV-1 RNA.</p></div
The ~80S assembly intermediate formed by WT Gag is the first assembly intermediate to contain unspliced viral RNA in human 293T cells and in the absence of PuroHS treatment.
<p><b>(A)</b> 293T cells transfected with the indicated construct (Set II construct in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006977#ppat.1006977.g001" target="_blank">Fig 1A</a>) were harvested following PuroHS treatment, and the number of unspliced viral RNA copies per 1000 cells was determined for cell lysate. (<b>B)</b> Lysate from A was analyzed by velocity sedimentation, and the number of unspliced viral RNA copies per 1000 cells was determined for each gradient fraction. (<b>C)</b> Gradient fractions from B were subjected to IP with αGFP, and the number of unspliced viral RNA copies per 1000 cells was determined for IP eluates from each gradient fraction. <b>(D)</b> COS-1 cells transfected to express WT Gag GFP and the V1B genome (Set II constructs in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006977#ppat.1006977.g001" target="_blank">Fig 1A</a>) were harvested under standard conditions without PuroHS treatment, and the number of unspliced viral RNA copies per 1000 cells was determined for cell lysates. (<b>E)</b> Lysate from D was analyzed by velocity sedimentation, and the number of unspliced HIV-1 RNA copies in the equivalent of 1000 cells was determined in each gradient fraction. <b>(F)</b> Gradient fractions from E were subjected to IP with αGFP, and the number of unspliced HIV-1 RNA copies in the equivalent of 1000 cells was determined for IP eluates from each fraction. Positions of kD markers are shown to the right of blots. Brackets at top show S value markers, and dotted lines demarcate assembly intermediates based on their migrations in the corresponding Gag WB. Error bars show SEM from duplicate samples. Data in each column are from a single experiment that is representative of two independent replicate experiments.</p
Gag-ABCE1 colocalization <i>in situ</i> upon provirus expression.
<p>(<b>A)</b> PLA was used to detect regions in which Gag is within 40 nm from ABCE1 <i>in situ</i>, with concurrent αGag IF for quantification of intracellular Gag levels. An experimental schematic for PLA methods is shown. For PLA experiments, 293T cells were transfected with the proviruses shown in diagram (Set 1 constructs in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006977#ppat.1006977.g001" target="_blank">Fig 1A</a>). <b>(B)</b> The average number of PLA spots per cell was determined for all Gag-positive cells in five randomly chosen fields, and normalized to Gag levels. <b>C)</b> Representative images. From left to right for each construct: Gag IF (green) with DAPI-stained nuclei (blue), Gag-ABCE1 PLA signal (red) with DAPI-stained nuclei (blue), and a merge of all three. Merge demonstrates that PLA spots are mainly in Gag-expressing cells. Inset in PLA panel shows a high magnification view of the cell to the left of the inset. Scale bars, 5 μm. Data are representative of three independent replicate experiments. Error bars show SEM (n = 5 fields). + indicates a significant difference relative to WT (p≤0.01).</p
Analysis of unspliced viral RNA in the ~80S and ~500S assembly intermediates from chronically infected H9 T cells.
<p><b>(A)</b> Human H9 T cells chronically infected with the indicated provirus (Set III construct in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006977#ppat.1006977.g001" target="_blank">Fig 1A</a>) were harvested following PuroHS treatment, and the number of unspliced HIV-1 RNA copies per 1000 cells was determined for cell lysates. <b>(B)</b> Lysate from A was also analyzed by velocity sedimentation, and the number of unspliced HIV-1 RNA copies per 1000 cells was determined for each gradient fraction. (<b>C)</b> Gradient fractions from B were subjected to IP with αABCE1 and the number of unspliced HIV-1 RNA copies per 1000 cells was determined for IP eluates from each gradient fraction. Positions of kD markers are shown to the right of blots. Brackets at the top show S value markers, and dotted lines demarcate assembly intermediates based on their migrations in the corresponding Gag WB. Error bars show SEM from duplicate samples. Data in each column are from a single experiment that is representative of three independent replicate experiments.</p
Non-translating unspliced HIV-1 RNA is primarily in diverse complexes ≥30S in the absence of assembling Gag.
<p><b>(A-E)</b> Lysate from COS-1 cells transfected with the assembly-incompetent MACA provirus (Set I constructs in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006977#ppat.1006977.g001" target="_blank">Fig 1A</a>) was divided into two pools that were either untreated (ribosomes intact) or treated with PuroHS (ribosomes dissociated). Both pools were analyzed in parallel by velocity sedimentation followed by RT-qPCR of paired gradient fractions using the appropriate qPCR primer sets to determine copy number of the indicated RNA (28S rRNA, 7SL RNA, unspliced HIV-1 RNA, HIV-1 Tat mRNA, or GAPDH mRNA). Quantity of the indicated RNA in gradient fractions is expressed as a distribution (% of RNA in all fractions) to allow comparison between different species of RNA. Blot in panel C shows migration of MACA protein, which is also graphed as a gray dotted line in C. Position of kD marker is shown to the right of the blot. Brackets at top show expected S value migrations, and horizontal bars show expected migrations of various ribonucleoprotein complexes. Error bars show SEM from duplicate samples. Data are from a single experiment that is representative of three independent replicate experiments.</p
The ~80S assembly intermediate contains Gag G2A associated with unspliced HIV-1 RNA while the ~10S Gag G2A assembly intermediate does not.
<p><b>(A)</b> COS-1 cells transfected with indicated MACA and G2A proviruses (Set I constructs in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006977#ppat.1006977.g001" target="_blank">Fig 1A</a>) were harvested following PuroHS treatment, and the number of unspliced HIV-1 RNA copies was determined for cell lysates. (<b>B)</b> Lysates from A were analyzed by velocity sedimentation, and the number of unspliced HIV-1 RNA copies per 1000 cells was determined for each fraction and normalized to the inputs shown in A. (<b>C)</b> Gradient fractions from B were subjected to IP with human polyclonal HIV immune globulin (αGag), and the number of unspliced HIV-1 RNA copies per 1000 cells was determined for IP eluates from each gradient fraction and normalized to the inputs shown in A. Similar results were obtained upon IP with monoclonal antibody to p24 but are not presented. (<b>D)</b> COS-1 cells transfected with the indicated Gag G2A provirus (Set 1 constructs in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006977#ppat.1006977.g001" target="_blank">Fig 1A</a>) were harvested following PuroHS treatment, and the number of unspliced HIV-1 RNA copies per 1000 cells was determined for cell lysate. (<b>E)</b> Lysate from D was also analyzed by velocity sedimentation, and viral RNA copy number per 1000 cells was determined for each gradient fraction. (<b>F)</b> Gradient fractions from E were subjected to IP with αDDX6, and the number of unspliced HIV-1 RNA copies per 1000 cells was determined for IP eluates from each gradient fraction. Positions of kD markers are shown to the right of blots. Brackets at top show S value markers, and dotted lines demarcate assembly intermediates based on their migrations in the corresponding Gag WB. Error bars show SEM from duplicate samples. Data in each column are from a single experiment that is representative of three independent replicate experiments.</p
The ~80S assembly intermediate formed by WT Gag and two Gag mutants is the first intermediate to contain unspliced viral RNA.
<p><b>(A)</b> COS-1 cells transfected with the indicated WT and Gag G2A constructs (Set II constructs in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006977#ppat.1006977.g001" target="_blank">Fig 1A</a>) were harvested following PuroHS treatment, and the number of unspliced viral RNA copies per 1000 cells was determined for cell lysates. (<b>B)</b> Lysates from A were analyzed by velocity sedimentation, and the number of unspliced viral RNA copies per 1000 cells was determined for each gradient fraction and normalized to inputs in A. (<b>C)</b> Gradient fractions from B were subjected to IP with αGFP, and the number of unspliced HIV-1 RNA copies per 1000 cells was determined for IP eluates from each gradient fraction and normalized to inputs in A. (<b>D)</b> COS-1 cells transfected with the indicated Gag W184A/M185A construct (Set II construct in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006977#ppat.1006977.g001" target="_blank">Fig 1A</a>) were harvested following PuroHS treatment, and the number of unspliced viral RNA copies per 1000 cells was determined for cell lysate. (<b>E)</b> Lysate from D was also analyzed by velocity sedimentation, and the number of unspliced viral RNA copies per 1000 cells was determined for each gradient fraction and normalized to inputs in A. (<b>F)</b> Gradient fractions from E were subjected to IP with αGFP and the number of unspliced viral RNA copies per 1000 cells was determined for IP eluates from each gradient fraction and normalized to inputs in A, with units indicated on left side Y-axis. Gradient fractions were also directly analyzed for 18S rRNA, to mark the position of the 40S small ribosomal subunit. Quantity of 18S rRNA in gradient fractions is expressed as a distribution (% of RNA in all fractions), with units indicated on the right side Y-axis. Positions of kD markers are shown to the right of blots. Brackets at top show S value markers, and dotted lines demarcate assembly intermediates based on their migrations in the corresponding Gag WB. Error bars show SEM from duplicate samples. Data in each column are from a single experiment that is representative of three independent replicate experiments.</p
Gag-DDX6 colocalization <i>in situ</i> upon provirus expression.
<p>(<b>A)</b> PLA was used to detect regions in which Gag is within 40 nm from DDX6 <i>in situ</i>, with concurrent αGag IF for quantification of intracellular Gag levels. An experimental schematic for PLA methods is shown. For PLA experiments, 293T cells were transfected with the proviruses shown in diagram (Set 1 constructs in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006977#ppat.1006977.g001" target="_blank">Fig 1A</a>). <b>(B)</b> The average number of PLA spots per cell was determined for all Gag-positive cells in five randomly chosen fields, and normalized to Gag levels. <b>C)</b> Representative images. From left to right for each construct: Gag IF (green) with DAPI-stained nuclei (blue), Gag-DDX6 PLA signal (red) with DAPI-stained nuclei (blue), and a merge of all three. Merge demonstrates that PLA spots are mainly in Gag-expressing cells. Inset in PLA panel shows a high magnification view of the cell to the right of the inset. Scale bars, 5 μm. Data are representative of three independent replicate experiments. Error bars show SEM (n = 5 fields). +++ indicates a significant difference relative to WT (p≤0.001).</p
The association of DDX6 with unspliced viral RNA is observed <i>in situ</i> at PM assembly sites for WT Gag but not for Gag Zip.
<p><b>(A)</b> HeLa cells expressing MCP-GFP were transfected with V1B genomes that contain MS2 binding sites (Set IV constructs in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006977#ppat.1006977.g001" target="_blank">Fig 1A</a>) and express WT Gag (shown), Gag Zip, or MACA. Cells were analyzed by double-label IEM, using αDDX6 and αGFP, which allowed detection of DDX6 (large gold) and MCP-GFP-labeled unspliced viral RNA (small gold), respectively. All early and late assembly sites at the PM were identified in ten cells (~250 μm of PM total per group), and scored for labeling of unspliced viral RNA, DDX6, and double labeling. (<b>B)</b> Graph shows the percentage of all early and late PM assembly events that display DDX6 labeling (total DDX6+), labeling of unspliced viral RNA (total viral RNA +), or double labeling (viral RNA+/DDX6+). (<b>C)</b> Images show representative assembly sites at the PM for each group, with symbols indicating early or late PM assembly sites that are single-labeled for either unspliced viral RNA or DDX6 (dark arrows), double-labeled (asterisks), or unlabeled (open arrows). Dotted lines outline the PM. Scale bars, 200 nm. Error bars show SEM (n = 10 cells). ++ indicates a significant difference between WT Gag and Gag Zip (p<0.005). For additional data, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006977#ppat.1006977.s009" target="_blank">S1 Table</a>.</p
Gag Zip fails to associate with viral-RNA-containing granules, but is rescued by a heterologous viral-RNA-binding domain.
<p><b>(A)</b> COS-1 cells transfected with indicated constructs (Set II constructs in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006977#ppat.1006977.g001" target="_blank">Fig 1A</a>) were harvested following PuroHS treatment, and the number of unspliced viral RNA copies per 1000 cells was determined for cell lysates. (<b>B)</b> Lysates from A were analyzed by velocity sedimentation, and the number of unspliced HIV-1 RNA copies per 1000 cells was determined for each gradient fraction and normalized to inputs in A. (<b>C)</b> Gradient fractions from B were subjected to IP with αGFP, and the number of unspliced viral RNA copies per 1000 cells was determined was determined for IP eluates from each gradient fraction and normalized to input in A. (<b>D)</b> COS-1 cells transfected with indicated constructs (Set II constructs in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006977#ppat.1006977.g001" target="_blank">Fig 1A</a>) were harvested following PuroHS treatment, and the number of unspliced viral RNA copies per 1000 cells was determined for cell lysates. (<b>E)</b> Lysates from D were analyzed by velocity sedimentation, and the number of unspliced viral RNA copies per 1000 cells was determined for each gradient fraction and normalized to inputs in A. (<b>F)</b> Gradient fractions from E were subjected to IP with αGFP, and the number of unspliced viral RNA copies per 1000 cells was determined for IP eluates from each gradient fraction and normalized to inputs in A. Positions of kD markers are shown to the right of blots. Brackets at top show S value markers, and dotted lines demarcate assembly intermediates based on their migrations in the corresponding Gag WB. Error bars show SEM from duplicate samples. Data in each column are from a single experiment that is representative of two independent replicate experiments.</p