The gp41 cytoplasmic tail mediates retromer-dependent Golgi retrieval.

<p><b>A)</b> Immunostaining of intracellular CD8-gp41CT (green) and Vps26 (red) in HeLa cells. Panels are single <i>xy</i> slices and are a representative example from three independent experiments. Coincident staining appears yellow. Scale bar is 10 microns. <b>B)</b> CD8-gp41CT expressing cells were treated with control or Vps26 siRNA. Cells were incubated with anti-CD8 monoclonal antibody at 4°C for 30 min, washed and incubated 37°C for 10 and 120 min. Cells were fixed, permeabilized and stained for the Golgi marker giantin (red) and fluorescently conjugated anti-mouse secondary antibody to localize the pool of internalized CD8-gp41CT (green). Panels are maximum intensity projections reconstructed from serial <i>Z</i> sections through the entire volume of the cell. Data are representative of three independent experiments. Scale bar is 20 microns. <b>C)</b> Immunostaining of total intracellular CD8-gp41CT (green) and Vps35 (red) in HeLa cells. Panels are single <i>xy</i> slices and are a representative example from three independent experiments. Coincident staining appears yellow. Scale bar is 15 microns. <b>D)</b> CD8-gp41CT expressing cells were treated with control or Vps35 siRNA and antibody-feeding and Golgi retrieval performed as described in B. Data are representative of three independent experiments. Scale bar is 20 microns. Western blot confirming efficient knockdown of Vps35 in HeLa cells following transfection with control or Vps35 siRNA as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004518#ppat-1004518-g001" target="_blank">Figure 1</a>. Band intensities were quantified using ImageJ and the percentage of remaining Vps35 relative to tubulin was calculated. One representative Western blot from three independent experiments is shown.</p

Immunofluorescence staining of Env in HIV-1 infected cells.

<p><b>A)</b> HeLa TZM-bl cells were infected with HIV-1, fixed, permeabilized and stained for HIV-1 Env (green) and retromer component Vps26 or the Golgi marker giantin or (red). Panels are single <i>xy</i> slices and are representative examples from three independent experiments. Arrows highlight coincident labeling of Env and Vps26. Scale bar is 20 microns. The amount of immunoreactive Env colocalizing with Vps26 or giantin was calculated from at least 20 cells.</p

Retromer Regulates HIV-1 Envelope Glycoprotein Trafficking and Incorporation into Virions

<div><p>The envelope glycoprotein (Env) of the Human Immunodeficiency Virus Type-1 (HIV-1) is a critical determinant of viral infectivity, tropism and is the main target for humoral immunity; however, little is known about the cellular machinery that directs Env trafficking and its incorporation into nascent virions. Here we identify the mammalian retromer complex as a novel and important cellular factor regulating Env trafficking. Retromer mediates endosomal sorting and is most closely associated with endosome-to-Golgi transport. Consistent with this function, inactivating retromer using RNAi targeting the cargo selective trimer complex inhibited retrograde trafficking of endocytosed Env to the Golgi. Notably, in HIV-1 infected cells, inactivating retromer modulated plasma membrane expression of Env, along with Env incorporation into virions and particle infectivity. Mutagenesis studies coupled with coimmunoprecipitations revealed that retromer-mediated trafficking requires the Env cytoplasmic tail that we show binds directly to retromer components Vps35 and Vps26. Taken together these results provide novel insight into regulation of HIV-1 Env trafficking and infectious HIV-1 morphogenesis and show for the first time a role for retromer in the late-steps of viral replication and assembly of a virus.</p></div

Identification of two internal sequences within the EnvCT required for retromer binding.

<p><b>A)</b> Antibody-feeding and retrieval assay was performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004518#ppat-1004518-g004" target="_blank">Figure 4</a>. Panels are maximum intensity projections reconstructed from serial <i>Z</i> sections through the entire volume of the cell. Data are representative of two independent experiments. Scale bar is 20 microns. <b>B)</b> Cell lysates prepared from cells stably expressing CD8-gp41CT constructs were incubated with anti-CD8 coated beads and co-IP proteins were subjected to SDS-PAGE and Western blotting for Vps35 and Vps26. Deleting either of two internal regions of the EnvCT abrogates co-IP of Vps26 and Vps35.</p

The C terminal 100 amino acids of the Env cytoplasmic tail are required to bind retromer.

<p><b>A)</b> Cells expressing reporter constructs containing truncated EnvCT were treated with control or Vps26 siRNA and an antibody feeding assay was performed to follow Golgi retrieval of endocytosed protein as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004518#ppat-1004518-g004" target="_blank">Figure 4</a>. Golgi marker giantin (red) and internalized CD8-gp41CT (green). Panels are maximum intensity projections reconstructed from serial <i>Z</i> sections through the entire volume of the cell. Data are representative of three independent experiments. Scale bar is 20 microns. <b>B)</b> Cells from A stained for Vps26 show that full length and truncated mutants are all efficiently endocytosed and colocalize with intracellular Vps26. Scale bar is 20 microns. <b>C)</b> Truncation of the gp41CT abrogates co-IP of Vps26 and Vps35. Cell lysates prepared from untransfected (UT) or cells stably expressing CD8-reporter constructs were incubated with anti-CD8 coated beads and co-IP proteins were subjected to SDS-PAGE and Western blotting for Vps35 and Vps26. Untransfected HeLa cells were used as a negative control and CD8-CIMPR as a positive control. <b>D)</b> HeLa cells expressing CD8-reporter constructs were treated with cycloheximide for the indicated periods of time and cell lysates subjected to SDS-PAGE and western blotting. One representative of two independent experiments is shown.</p

Vps26 depletion increases plasma membrane expression of HIV-1 Env and incorporation into viral particles.

<p><b>A)</b> HeLa TZM-bl cells were transfected twice with control or Vps26 siRNA. Forty-eight hours after the second transfection cell lysates were prepared and subjected to SDS-PAGE and western blotting for Vps26 and actin as a loading control. Band intensities were quantified using ImageJ and the percentage of remaining Vps26 relative to actin was calculated. One representative Western blot from three independent experiments is shown. <b>B)</b> HeLa TZM-bl cells were infected with HIV-1 immediately after the second knockdown. Forty-eight hours later viral supernatants were pelleted by ultracentrifugation through sucrose and subjected to SDS-PAGE and western blotting for HIV-1 Env (gp120 and gp41) and Gag (p24CA). One representative Western blot from three independent experiments is shown. The band intensities from three independent experiments were quantified using ImageJ and the ratio of HIV-1 Env gp120 to Gag p24CA is shown. Error bars show the SEM. Cell lysates were also prepared and blotted for Gag p55 (lower panel). <b>C)</b> Equal volumes of viral supernatants from infected cells were used to infect Jurkat 1G5 reporter cells. Infectious virus titer is shown as luciferase relative light units (RLU). One representative of three independent experiments is shown. Errors bars show the SEM. <b>D)</b> Flow cytometry analysis of HIV-1 Env plasma membrane expression following Vps26 KD. One histogram representative of three independent experiments is shown. Cells were either left uninfected (filled grey), untreated and infected (solid grey line), control siRNA treated and infected (broken black line) or Vps26 siRNA treated and infected (solid black line) and surface stained for Env on ice prior to fixation. <b>E)</b> Pooled cell surface mean fluorescence intensity (MFI) of Env staining from three independent experiments. Error bars show the SEM. <b>F)</b> Cell lysates from HIV-1 infected siRNA treated cells were subjected to SDS-PAGE and Western blotting for Vps26, tubulin, HIV-1 Env and Gag. One representative of three independent experiments is shown. The band intensities from three independent experiments were quantified using ImageJ and the ratio of unprocessed (gp160) and processed (gp120) Env and Gag (p55) is shown. Error bars show the SEM.</p

Prediction of the time to AIDS, <i>t</i>_<i>A</i>, for different treatment schemes.

<p>We consider 30-day treatments starting <b>(A)</b> on the 3rd day and <b>(B)</b> when <i>N</i> = 500 <i>cells</i>/<i>μl</i>, respectively. All treatments block cell-free infection completely (<i>β</i>₂ = 0 and <i>g</i> = 0). The treatments also affect cell-to-cell infection (x axis) and / or manipulate the CD4⁺ T cell activation process (y axis, see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004179#pcbi.1004179.g004" target="_blank">Fig. 4A</a>). The black squares represent treatments that reduced the density of infected cells, latent cells and free virions all below 10⁻¹² per <i>μl</i>.</p

Impact of treatment starting time on HIV progression.

<p>HIV progression for a 30-day ‘perfect’ treatment starting at three different times after the initial infection: (1) on the 3rd day when the density of all CD4⁺ T cells is <i>N</i> = 725 <i>cells</i>/<i>μl</i>, (2) when <i>N</i> = 500 <i>cells</i>/<i>μl</i>; (3) when <i>N</i> = 350 <i>cells</i>/<i>μl</i>. The ‘prefect’ treatment here means both cell-to-cell infection and the cell-free infection are completely blocked (i.e. <i>β</i>₁ = 0, <i>β</i>₂ = 0 and the virus release rate <i>g</i> = 0) for 30 days.</p

The HIV-1 model reproduces the full course of HIV-1 infection.

<p><b>(A)</b> Diagrammatic representation of the model described by <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004179#pcbi.1004179.e001" target="_blank">Equation 1</a>. <b>(B)</b> Numerical solutions of the model, plotting <i>N</i>, the density of all CD4⁺ T cells (y axis on the left) and <i>V</i>, the density of free virions (y axis on the right in log scale) as a function of time (in days or weeks), respectively. The initial infection starts on day 0 and the cellular immune response starts on day 30. Parameter values are in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004179#pcbi.1004179.t001" target="_blank">Table 1</a>.<b>(C)</b> The density of quiescent, susceptible, latent and infected CD4⁺ T cells, and the density of free virions as function of time (in days).</p

Two modes of HIV-1 infection.

<p>The density of CD4⁺ T cells as a function of time for different values of cell-to-cell infection rate <i>β</i>₁ and cell-free infection rate <i>β</i>₂: (1) both use their default value, (2) <i>β</i>₁ uses its default value and <i>β</i>₂ = 0, (3) <i>β</i>₁ = 0 and <i>β</i>₂ uses its default value, (4) <i>β</i>₁ is twice its default value and <i>β</i>₂ = 0, (5) <i>β</i>₁ = 0 and <i>β</i>₂ is twice its default value.</p