The host protein Staufen1 interacts with the Pr55zinc fingers and regulates HIV-1 assembly via its N-terminus-3

Ubulin-binding domain (TBD) are represented as grey and black boxes, respectively. Schematic representation of the Pr55/Pr55BRET assay. This assay is used as a sensor of Pr55multimerization. 293T cells were transfected with constant amounts of pCMV-Pr55-luc and increasing amounts of pCMV-Pr55-YFP. A constant amount of a third plasmid expressing Stau1-HAor Stau1-HAwas included in the transfection procedure. luc activity as well as transmitted and total YFP activities was measured. BRET ratios were plotted in function of their corresponding total YFP/luc ratio which allows us to compare BRET ratios at the same relative expression levels of Pr55fusion proteins. This figure is representative of four independent experiments. Cells corresponding to the four last points of each curve from Figure 4B were lysed. Cell lysates were analyzed by Western blotting using anti (α)-HA antibodies for their content in over-expressed Stau1 proteins. *: Non-specific labelling typically obtained with the anti-HA antibody. BRET ratios were compared at comparable total YFP/luc ratio. The BRET ratio corresponding to the pr55fusions expressed alone was arbitrarily set to 1. The BRET induction levels were then determined and are shown in the graph. These results are representative of 4 experiments.<p><b>Copyright information:</b></p><p>Taken from "The host protein Staufen1 interacts with the Pr55zinc fingers and regulates HIV-1 assembly via its N-terminus"</p><p>http://www.retrovirology.com/content/5/1/41</p><p>Retrovirology 2008;5():41-41.</p><p>Published online 22 May 2008</p><p>PMCID:PMC2409373.</p><p></p

The host protein Staufen1 interacts with the Pr55zinc fingers and regulates HIV-1 assembly via its N-terminus-6

Ach condition, an aliquot of the cells (providing equivalent total YFP/luc ratio) was used for Western blot analysis using anti (α)-HA and anti (α)-CA antibodies. Anti (α)-GAPDH antibody was used as a loading control. Another aliquot of the cells was used for BRET assays. Calculated BRET ratios were plotted as a function of the corresponding total YFP/luc ratio. Dose response pr55/pr55BRET assays. 293T cells were transfected with fixed amounts of pCMV-pr55-luc and pCMV-pr55-YFP and increasing amounts of different Stau1-HA expressors. 48 hours later, half of the cells were lysed and analyzed by Western blotting using anti (α)-HA and anti (α)-GAPDH antibodies. *: Non-specific labelling.The other half of the cells was used for BRET assays. BRET ratio is plotted as a function of the corresponding amount of transfected Stau1-HA expressor.<p><b>Copyright information:</b></p><p>Taken from "The host protein Staufen1 interacts with the Pr55zinc fingers and regulates HIV-1 assembly via its N-terminus"</p><p>http://www.retrovirology.com/content/5/1/41</p><p>Retrovirology 2008;5():41-41.</p><p>Published online 22 May 2008</p><p>PMCID:PMC2409373.</p><p></p

The host protein Staufen1 interacts with the Pr55zinc fingers and regulates HIV-1 assembly via its N-terminus-2

YFP-fused Gag proteins were expressed in 293T cells for twenty-four hours. VLPs in the cell supernatant were purified. Cell lysates and VLPs were analyzed by Western blotting using anti (α)-GFP antibodies. Anti (α)-GAPDH antibodies were used as loading controls. This figure is representative of three independent experiments. 293T cells were transfected with constant amounts of pCMV-Pr55-luc and increasing amounts of wild type or mutated YFP-fused Gag expressors. Twenty-four hours post-transfection, cells were collected and BRET ratios determined. BRET ratios are plotted in function of their corresponding total YFP/luc ratio. This figure is representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "The host protein Staufen1 interacts with the Pr55zinc fingers and regulates HIV-1 assembly via its N-terminus"</p><p>http://www.retrovirology.com/content/5/1/41</p><p>Retrovirology 2008;5():41-41.</p><p>Published online 22 May 2008</p><p>PMCID:PMC2409373.</p><p></p

The host protein Staufen1 interacts with the Pr55zinc fingers and regulates HIV-1 assembly via its N-terminus-4

P1-luc and Stau1-YFP expressors. 48 hours post-transfection, Stau1-YFP and Stau1-YFP contents in the cells were analyzed by Western blotting using anti (α)-GFP antibodies. 293T cells were transfected with constant amounts of CA-p2-NC-p1-Rluc-expressing plasmid and increasing amounts of wild-type or mutated YFP-fused Stau1 expressors. Twenty four hours post-transfection, live transfected cells were used for CA-p2-NC-p1/Stau1 BRET assays. BRET ratios are plotted in function of their corresponding total YFP/luc ratio. n = 4. BRET ratios were compared at identical total YFP/luc ratio and corrected by subtracting the background BRET ratio calculated for unfused YFP and CA-p2-NC-p1-luc co-expression. The corrected BRET ratio between CA-p2-NC-p1-luc and Stau1-YFP was arbitrarily set to 100%. n = 4. 293T cells were co-transfected with Pr55and wild-type or N-terminally truncated Stau1-Flag expressors. Twenty-four hours post-transfection, cell extracts were prepared and subjected to immunoprecipitation using anti-Flag antibodies. Cell lysates and immune complexes were analyzed by Western blotting using anti (α)-Flag and anti (α)-CA antibodies. Anti (α)-GAPDH antibodies were used as a loading control. n = 2.<p><b>Copyright information:</b></p><p>Taken from "The host protein Staufen1 interacts with the Pr55zinc fingers and regulates HIV-1 assembly via its N-terminus"</p><p>http://www.retrovirology.com/content/5/1/41</p><p>Retrovirology 2008;5():41-41.</p><p>Published online 22 May 2008</p><p>PMCID:PMC2409373.</p><p></p

The host protein Staufen1 interacts with the Pr55zinc fingers and regulates HIV-1 assembly via its N-terminus-8

Tations were introduced in the basic region or in the zinc fingers of NC-p1-YFP fusion protein to generate four mutants. 293T cells were transfected with YFP, NC-p1-YFP and mutated NC-p1-YFP expressors. 48 hours post-transfection, cell lysates were prepared and analyzed by Western blotting using anti (α)-GFP antibodies.<p><b>Copyright information:</b></p><p>Taken from "The host protein Staufen1 interacts with the Pr55zinc fingers and regulates HIV-1 assembly via its N-terminus"</p><p>http://www.retrovirology.com/content/5/1/41</p><p>Retrovirology 2008;5():41-41.</p><p>Published online 22 May 2008</p><p>PMCID:PMC2409373.</p><p></p

Characterization of selected NS1 mutants reveals specific defects in infectious particle production.

<p><b>(A)</b><i>Replication kinetics of NS1 mutants in the context of a sub-genomic replicon</i>. Schematic representation of the sub-genomic reporter replicon (sgDVR2A) is shown at the top. It is derived from the DV2 full-length genome by insertion of a <i>Renilla luciferase</i> coding sequence in-between the capsid cyclization sequence (CAE) and the <i>Tosea asigna</i> virus 2A cleavage site. The last 24 amino acid residues of the envelope coding region (TM) at the N-terminus of NS1 ensure proper membrane topology of the polyprotein after synthesis. Selected NS1 mutations affecting virus production were inserted into sgDVR2A, and <i>in vitro</i> transcribed RNAs were electroporated into VeroE6 cells. Luciferase activity was measured in the lysates 4, 24, 48 and 72 h later. Values are expressed as fold of the 4h-value which reflects transfection efficiency. The background of the assay is determined with the active site NS5 polymerase mutant (GND). Columns represent mean and standard deviations of three independent experiments. (<b>B</b>) <i>Intra- and extra-cellular infectivity titers of NS1 mutants</i>. The structure of the full-length DENV genome used for this analysis is shown at the top (DV). NS1 mutations specified at the bottom were inserted into DV and in vitro transcripts derived therefrom were transfected into Huh7 cells and used for determination of intra- and extracellular infectivity. Seventy-two hours post electroporation, supernatants and cell lysates were subjected to 3 freeze and thaw cycles and titers were determined by TCID₅₀ assay using Huh7 cells and an envelope protein-specific MAb. Mean values and standard deviations of three independent experiments are shown. (<b>C</b>) Evidence that NS1 mutations affect, in part, release of infectious DENV particles. To determine the relative contribution of defects in virus assembly and virus release, data presented in (B) were used to calculate the ratio of intra- vs. extra-cellular infectivity.</p

Effect of mutations in NS1 on the production of infectious DENV particles.

<p>(<b>A</b>) <i>Released infectivity from DVR2A NS1 mutant-transfected cells</i>. VeroE6 cells were electroporated with DENV genomes containing NS1 mutations specified at the bottom. Seventy-two hours post-electroporation, supernatants containing infectious virus were used to infect naïve VeroE6 cells and luciferase activity measured 48 h later (black columns). Luciferase activity in the lysates 72 h post-electroporation reflecting replication is also shown (white columns). The background of the assay is determined with the active site NS5 polymerase mutant (GND). Mean values and standard deviations of three independent experiments are shown. Colours at the top of the panels refer to NS1 protein domains as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005277#ppat.1005277.g002" target="_blank">Fig 2A</a>. (<b>B</b>) <i>Efficiency of virus production relative to replication fitness</i>. To calculate specific defects in infectious particle production, data presented in (A) are shown as ratio of released infectivity/viral replication, and expressed as fold of wild-type (WT). Note that only NS1 mutants with viral RNA replication above input values were considered (cf. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005277#ppat.1005277.g002" target="_blank">Fig 2B</a>; replication > Log₁₀0).</p

NS1 interacts with the structural proteins.

<p>(<b>A</b>) <i>Capsid</i>, <i>prM and Envelope proteins co-immunoprecipitate with NS1</i>. Naïve VeroE6 (CTRL), VeroE6_NS1^WT (WT) or VeroE6_NS1^HA (HA) cells were infected with 1 MOI of DVR2A^ΔNS1 TCPs. Forty-height hours post-infection, cell lysates clarified by centrifugation were used for immunoprecipitation with HA-affinity agarose beads and eluates (IP) or whole cell lysates (Input) analyzed by western-blotting using antibodies specified on the right of each panel. Numbers on the left refer to molecular weight standards expressed in kDa; black arrowhead on the right indicates the ΔNS1 protein expressed by the DVR2A^ΔNS1 genome. A representative experiment of four independent repetitions is shown. (<b>B</b>) <i>Interaction between NS1 and prM/E in DENV-2 wild-type virus-infected cells</i>. VeroE6 cells were mock infected or infected with DENV-2 at an MOI of 1. Forty-eight hours later clarified cell lysates were used for immunoprecipitation using a NS1-specific rabbit polyclonal antiserum or the corresponding pre-immune (PIS) antiserum and protein A-Sepharose beads. After extensive washing, eluted protein complexes were analyzed by western-blotting using polyclonal anti-NS1 and anti-prM or mouse monoclonal anti-E or anti-C specific antibodies as specified on the right of each panel. DENV proteins are indicated with arrowheads, asterisks refer to the immunoglobulin heavy chain.</p

Effect of NS1 Alanine substitutions on DENV replication.

<p>(<b>A</b>) <i>Schematic representation of the DENV reporter virus genome and NS1 protein domains</i>. The full-length luciferase reporter DENV genome (DVR2A) is shown at the top, with the 5’ and 3’ NTRs depicted with their putative secondary structures. Polyprotein cleavage products are separated by vertical lines and labeled as specified in the introduction. A <i>Renilla luciferase</i> coding sequence was inserted in-between the capsid cyclization sequence (CAE) and the <i>Tosea asigna</i> virus 2A cleavage site that ensures proper processing after polyprotein synthesis. NS1 protein domains shown below are indicated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005277#ppat.1005277.g001" target="_blank">Fig 1</a>. Glycosylation sites are shown with green hexagons, while the <i>β-roll</i>, <i>Wing</i> and <i>β-ladder</i> domains are shown in blue, yellow and red, respectively. Two <i>connector</i> sub-domains within the <i>Wing</i> domain are shown in orange. (<b>B</b>) <i>Replication kinetics of DENV NS1 mutants</i>. VeroE6 cells were electroporated with <i>in-vitro</i> transcribed luciferase virus RNAs containing NS1 mutations specified at the bottom. Cells were lysed 4, 24, 48, 72, 96 and 120 h after transfection and luciferase activity in cell lysates was determined. Data were normalized to the 4h-value that reflects transfection efficiency. The background of the assay was determined with the active site NS5 polymerase mutant (GND). Colored lines on the top of each panel refer to the color-coded representation of NS1 protein domains as shown in (A). NS1 mutants severely impaired in viral RNA replication (normalized luciferase activity ≤0) are underlined. For ease of comparison, data obtained with WT and GND are repeated in the left of each panel. Mean values and standard deviations of three independent experiments are shown.</p

C-terminally mCherry-tagged NS1 supports DENV RNA replication and virus production and co-localizes with envelope and capsid upon ΔNS1^TCP infection.

<p>(<b>A</b>) <i>Sub-cellular distribution of NS1</i>^{<i>mCherry</i>}. Two days post-cell seeding, CTRL, NS1^WT- or NS1^mCherry-expressing cells were fixed, permeabilized and NS1 was detected with a rabbit polyclonal NS1-specific primary and AlexaFluor-488-conjugated secondary antibody. Cells were analyzed by confocal microscopy as described in materials and methods. The upper panel depicts a schematic representation of the NS1^mCherry fusion protein. (<b>B</b>) <i>Replication competence of ΔNS1</i>^<i>TCP</i><i>in VeroE6_NS1</i>^{<i>mCherry</i>}<i>cells</i>. VeroE6 cells expressing no NS1 (CTRL) or NS1^WT or NS1^mCherry were infected with 1 MOI of ΔNS1^TCP and luciferase activity was measured in the lysates 24, 48 and 72 h post-infection. Released infectivity was assessed by inoculating NS1^WT-expressing cells with culture media collected 72 h p.i. and measuring luciferase activity 48 h later. The dashed lines indicate the limit of detection of the assay. (<b>C-D</b>) <i>Co-localization and 3D reconstruction of NS1 with the structural proteins capsid and envelope</i>. (C) VeroE6 cells specified on the left, were mock-infected (Non-Infected) or infected with 1 MOI of ΔNS1^TCP. Two-days later, cells were fixed, permeabilized and stained with E- and capsid-specific antibodies. Cells were analyzed by confocal microscopy. Scale bars represent 10 μm. (D) Deconvolved fluorescence image of VeroE6_NS1^mCherry cells infected with ΔNS1^TCP particles. VeroE6 cells stably expressing C-terminally tagged NS1^mCherry were infected with ΔNS1^TCP as described above. Two-days later, cells were fixed, permeabilized and stained with E- and capsid-specific antibodies. <i>Left panel</i>: representative image of an NS1^mCherry- ΔNS1^TCP-infected cell 48 h p.i. (scale bar represents 2 μm). Boxed areas are enlarged in the numbered panels on the right (scale bar represent 300 nm). Overview and enlargements show 3D reconstructed images created with the Imaris software as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005277#sec010" target="_blank">Materials and Methods</a>.</p