Suppression of Foxo1 Activity and Down-Modulation of CD62L (L-Selectin) in HIV-1 Infected Resting CD4 T Cells

<div><p>HIV-1 hijacks and disrupts many processes in the cells it infects in order to suppress antiviral immunity and to facilitate its replication. Resting CD4 T cells are important early targets of HIV-1 infection in which HIV-1 must overcome intrinsic barriers to viral replication. Although resting CD4 T cells are refractory to infection <i>in vitro</i>, local environmental factors within lymphoid and mucosal tissues such as cytokines facilitate viral replication while maintaining the resting state. These factors can be utilized <i>in vitro</i> to study HIV-1 replication in resting CD4 T cells. <i>In vivo</i>, the migration of resting naïve and central memory T cells into lymphoid tissues is dependent upon expression of CD62L (L-selectin), a receptor that is subsequently down-modulated following T cell activation. CD62L gene transcription is maintained in resting T cells by Foxo1 and KLF2, transcription factors that maintain T cell quiescence and which regulate additional cellular processes including survival, migration, and differentiation. Here we report that HIV-1 down-modulates CD62L in productively infected naïve and memory resting CD4 T cells while suppressing Foxo1 activity and the expression of KLF2 mRNA. Partial T cell activation was further evident as an increase in CD69 expression. Several other Foxo1- and KLF2-regulated mRNA were increased or decreased in productively infected CD4 T cells, including IL-7rα, Myc, CCR5, Fam65b, S1P₁ (EDG1), CD52, Cyclin D2 and p21^CIP1, indicating a profound reprogramming of these cells. The Foxo1 inhibitor AS1842856 accelerated <i>de novo</i> viral gene expression and the sequella of infection, supporting the notion that HIV-1 suppression of Foxo1 activity may be a strategy to promote replication in resting CD4 T cells. As Foxo1 is an investigative cancer therapy target, the development of Foxo1 interventions may assist the quest to specifically suppress or activate HIV-1 replication <i>in vivo</i>.</p></div

Diagram of software architecture of HIVToolbox.

<p>Diagram of software architecture of HIVToolbox.</p

Sources of data in the HIVToolbox MySQL database.

<p>*Sequence features that are multimerization interfaces were calculated in Molmol based on residues that were less than 3.25 Å away from at least one residue in another subunit <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020122#pone.0020122-Koradi1" target="_blank">[63]</a>.</p

A model of IN:LEDGF:viral DNA based on the PFV IN structure.

<p>(<b>A</b>) A model of HIV-1 IN complex with 4 IN subunits, 4 LEDGF subunits (light gray), and two viral DNA strands (dark gray); (<b>A, B</b>) Left panels (IN NTD = green, CCD = blue, CTD = red. (<b>A, B</b>) Right panels show functional sites (green = DNA binding), red = dimerization interface in 1EX4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020122#pone.0020122-Chen1" target="_blank">[10]</a>, cyan = dimerization interface in 1WJA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020122#pone.0020122-Cai1" target="_blank">[11]</a>; blue = tetramerization interface in 1K6Y <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020122#pone.0020122-Wang1" target="_blank">[8]</a>; purple = zinc binding site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020122#pone.0020122-Cai1" target="_blank">[11]</a>; brown = reverse transcriptase binding site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020122#pone.0020122-Wilkinson1" target="_blank">[31]</a>, light brown = tetramerization residues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020122#pone.0020122-Lutzke2" target="_blank">[16]</a>. (<b>B</b>) A 90° rotation about the Y-axis of <b>A</b>. Orange arrowhead indicated channel proposed to bind target DNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020122#pone.0020122-Hare1" target="_blank">[36]</a>.</p

Analysis of Integrase model tetramers and hetero-octamers.

<p>Output of HIVToolbox showing surface plots of IN structural models. (<b>A, B</b>) IN tetramers showing domain organization (left panels) and locations of actives site residues (royal blue), proposed viral DNA binding grooves (yellow lines), proposed genomic DNA binding channel (red line), and zinc binding sites (cyan). Yellow numbers indicate the subunit too which the domain belongs. (<b>C</b>) IN:LEDGF hetero-octamers models showing organization of proteins (left panel) and proposed DNA binding groove (middle panel, red line). LEDGF subunits are colored grey. (<b>D</b>) An end-on view of the proposed host DNA binding channel in the IN:LEGDF hetero-octamer model shown in (<b>C</b>) (red circle).</p

Sequence conservation of CK2 sites in different strains of HIV-1.

<p>*‘del’ indicate that the residue is deleted or was not present in one or more structures.</p

Foxo1 inhibitor AS1842856 accelerates HIV-1 expression.

<p>AS1842856 (AS) was applied to cells 1 day before and 1 day after infection with single round GFP reporter virus. CD45RO- naïve T cells are shown. <b>A.</b> GFP and CD62L expression in infected naïve CD4+ T cells on the indicated day after infection. Donor A is shown. <b>B.</b> The number of GFP+ cells on the indicated day after infection. Data are average and SD of the two donors. <b>C.</b> Mean fluorescence intensity (MFI) of GFP in the GFP+ cells. Data are average and SD of the two donors. <b>D.</b> Fold increase in the number and the MFI of GFP+ cells, and total GFP fluorescence in the cells calculated as the product of the number of GFP+ cells multiplied by their MFI. Data are average and SD of the two donors. <b>E.</b> Near-full length reverse transcripts on day 2 after infection for one representative cell donor. Data are averages and SD for replicate PCR reactions for each donor. <b>F.</b> HIV-1 fully spliced RNA presented relative to day 1 DMSO condition. Data are averages and SD for replicate PCR reactions for each donor. <b>G.</b> Virus production measured by genomic RNA in culture medium. Data are averages and SD for replicate PCR reactions for each donor. <b>H.</b> CD62L MFI on the indicated day. Data are MFI of GFP+ cells divided by background staining. Data are average and SD of the two donors. <b>I.</b> CD69 MFI on day 7 post infection on naïve and memory resting CD4+ T cells for donor A.</p

Interactive protein display page for Tat in HIVToolbox.

<p>Sequence window, Structure windows, Log windows, and Sequence Alignment section of HIVToolbox are shown. The interactive results page for HIV-1 tat is shown. The scrollable sequence window shows the protein sequence, domains (with colored fonts), functional residues (highlighted), protein-protein interaction sites (thin lines under sequence), mapped protein structures (thin colored lines over sequence) and minimotifs (figures under sequence). The synchronized interactive structural displays show domains and selected minimotifs (left panel), functional sites and selected protein-protein interaction sites (center panel), and residues conserved at or above a sequence conservation threshold selected with a slider or text box (right panel). The Sequence Alignment section shows alignment of a representative set of 20 sequences with the RefSeq sequence and the structure sequence. A second tab reveals a position specific-scoring matrix of amino acid frequencies at each position in the protein. More details about the features and use of HIVToolbox are in the supplement, and video tutorials are at Bio-Toolkit [<a href="http://www.bio-toolkit.com" target="_blank">http://www.bio-toolkit.com</a>].</p

Statistics for data in the HIVToolbox database.

<p>Statistics for data in the HIVToolbox database.</p

Analysis of Integrase with HIVToolbox.

<p>(<b>A–C</b>) Output of HIVToolbox showing relationships of IN elements shown in a model constructed from superposition of the catalytic domains in structures 1EX4 and 1K6Y. Residues 1–7, 47–55, 140–148, and 270–288 are unstructured and not shown. The left panels shows domains [NTD (red), CCD (Blue), and CTD (green)] and minimotifs; the center panels show functional sites and protein-protein interactions the right panels shows residues that are >98% conserved in 3787 HIV-1 IN isolates (yellow). (<b>A</b>) Location of three of the four putative CK2 phosphorylation sites located on the surface of the IN CCD (left panel); the 4^th CK2 site is in the CTD unstructured region. Numbers indicate the positions of putative phosphorylation sites. D270 is the last residue in the structure (orange). Conservation of the residues on CK2 sites is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020122#pone-0020122-t001" target="_blank"><b>Table 1</b></a>. (<b>B</b>) Conservation and location of the dimerization interface(s). Residues at the dimerization interface less than 3.25 Å from atoms in the other chain are colored: (red, 1EX4), (cyan, 1WJA), and (lighter cyan, 1K6Y). (<b>B, C</b>) Conservation and location of protein-protein interaction sites, modification sites, and DNA binding sites. (<b>C</b>) is a 180° rotation of (<b>B</b>) about the z-axis. (<b>A, B, C</b>) Sites are colored: DNA binding = green, Importin 7 binding = dark purple and dark green, Zn binding = purple, Karyopherin α5 binding = teal and orange, LEDGF binding = teal, Lysine acetylation = dark green, proline isomerization = orange, active site = royal blue, reverse transcriptase (RT) binding = brown.</p