Soluble factors from R5- or X4 tropic HIV-1 infected PBMCs activate human hepatic stellate cells.

<p>LX2 cells were treated with virus-free culture supernatants from HIV-1 infected (R5- or X4- tropic) human PBMCs for 72 hr. Mock (M) represents control group that was treated with supernatants from uninfected PBMCs. (A) Total RNA was isolated from LX2 cells and used for the expression analysis of various pro-fibrogenic and pro-inflammatory marker genes using qRT-PCR, as described in Methods. Data represents mean ± S.E. of three independent experiments. *p<0.05 and **p<0.01 vs mock. (B) LX2 cells were treated with culture supernatants from HIV-1 infected PBMCs for 24 hr as described in Methods. Total cell lysates were analyzed for TIMP-1 expression by western blotting. GAPDH was used as a loading control. Data representative of one of the three experiments is shown. (C) LX2 cells were plated in a 96-well plate at passage 4 and serum starved for 24 hr. The cells were then incubated for 72 hr with culture supernatants from X4-, R5- or mock- infected PBMCs. Following treatment, cells were assayed for proliferation and cell survival using the MTT assay. Data represents mean ± S.E. of three independent experiments.</p

Pathway analysis and validation in treated LX2 cells.

<p>Heat map showing predicted pathways for the miRNAs that were differentially modulated in LX2 cells treated with supernatants from (A) R5-tropic or (B) X4-tropic HIV-1 infected PBMCs. DIANA miRPath v2.0 was used to predict the pathways with default p-value threshold of 0.05 and microT threshold of 0.8. This clustering is based on significance analysis. All significantly targeted pathways are marked with deep red. On the vertical axis, miRNAs are clustered together by exhibiting similar pathway targeting patterns.</p

HIV-1 Infected Peripheral Blood Mononuclear Cells Modulate the Fibrogenic Activity of Hepatic Stellate Cells through Secreted TGF-β and JNK Signaling

<div><p>Background and Aims</p><p>Patients with liver disease infected with the human immunodeficiency virus (HIV) exhibit accelerated progression of hepatic fibrosis and liver cirrhosis compared to uninfected individuals. We studied the effects of soluble factors secreted by HIV-infected peripheral blood mononuclear cells (PBMCs) on hepatic stellate cells (HSCs), which are central mediators of liver fibrosis.</p><p>Methods</p><p>An <i>in vitro</i> model was used in which a HSC line, LX2, was treated with culture supernatants of human PBMCs infected with macrophage tropic (R5) or T cell tropic (X4) strains of HIV-1. Quantitative reverse transcription PCR (qRT-PCR) and western blotting were used to assess the expression of fibrogenic and proinflammatory markers; LX2 proliferation and intracellular signaling pathways were also studied. A qRT-PCR based miRNome array was used for comparative miRNA profiling of LX2 cells treated with infected PBMC culture supernatants.</p><p>Results</p><p>Pro-fibrogenic, angiogenic and proinflammatory markers, and proliferation of LX2 cells were increased following exposure to culture supernatants from HIV-1 infected PBMCs. The profiling of miRNAs in LX2 cells treated with culture supernatants from HIV-1 R5- or X4-infected PBMCs showed 66 and 22 miRNAs respectively, to be significantly altered compared to mock-treated LX2 cells. While different sets of miRNAs were altered in the two cases, bioinformatics analyses predicted these to be associated with common pathways, including TGF-β signaling and extracellular matrix receptor interaction pathways.</p><p>Conclusions</p><p>HIV infection creates a favorable milieu for the activation of hepatic stellate cells and increased hepatic fibrosis. We identify some regulatory molecules important for these effects.</p></div

Oligomerization of the human immunodeficiency virus type 1 (HIV-1) Vpu protein – a genetic, biochemical and biophysical analysis-1

<p><b>Copyright information:</b></p><p>Taken from "Oligomerization of the human immunodeficiency virus type 1 (HIV-1) Vpu protein – a genetic, biochemical and biophysical analysis"</p><p>http://www.virologyj.com/content/4/1/81</p><p>Virology Journal 2007;4():81-81.</p><p>Published online 29 Aug 2007</p><p>PMCID:PMC2042504.</p><p></p>g panels that in turn show transformants streaked in each section of the indicated plates. Growth is seen as light streaks on a dark background, except in the β-galactosidase filter assay where signal is seen as dark streaks on a light background. (B) Complete results for the entire screen using NL-Vpu and R5-Vpu full-length, transmembrane domain and cytoplasmic domain fusions to the Gal4 protein DNA-binding domain (BD) or activation domain (AD). Growth (+) or no growth (-) of transformants on various media is shown. LTH-3AT represents growth on SDLeuTrpHisplates containing 20 mM 3-amino-1,2,3-triazole. The β-galactosidase filter assay results are indicated as + or - and the liquid β-galactosidase assay values are shown in parentheses as an average of two independent measurements. Various negative and positive controls are also shown

Differentially regulated miRNAs in LX2 cells.

<p>qRT-PCR profiling array result showing fold upregulation or downregulation of the indicated miRNAs in LX2 cells treated with supernatants from (A) R5-tropic or (B) X4-tropic HIV-1 infected PBMCs as compared to mock-infected PBMCs. Fold changes were determined using comparative C_T method. Data represents mean of the three independent experiments. Only those miRNAs that were significantly modulated (p<0.05) in R5- or X4- supernatant treated LX2 cells, as compared to mock-treated cells, are shown.</p

miRNA expression profiling of LX2 cells.

<p>LX2 cells were treated with virus-free culture supernatants from mock, R5- or X4-tropic HIV-1 infected PBMCs for 72 hr. Total RNA was isolated and subjected to miRNA profiling as described in Methods. (A) Percent distribution of C_T values for the 1008 miRNAs across the samples. Any C_T value above 35 was taken as 35. Entire profiling was done in set of three and data represents mean ± S.E. of three independent experiments. (B) Heat map depicting relative expression levels of miRNAs that showed significant changes across all the biological replicates. The heat map shows hierarchical clustering of miRNAs. Each row represents miRNA and each column represents samples tested. Red and green represent miRNAs with expression levels above and below the mean, respectively.</p

Pore profile.

<p>(A) View along the pore axis from the C-terminal showing the Ser23 residue in “licorice” representation. Serine faces the interior of the channel in the pentamer model. (B) Side view of the pentamer model showing the location of the Ser23 residue (in “licorice” representation) and water molecules in the pore. The N-terminal side is on the top and the C-terminal is at the bottom. (C) Pore radius across the axis of the pentamer model. The pore is constricted towards the N-terminal side (top half).</p

Oligomerization of the human immunodeficiency virus type 1 (HIV-1) Vpu protein – a genetic, biochemical and biophysical analysis-4

<p><b>Copyright information:</b></p><p>Taken from "Oligomerization of the human immunodeficiency virus type 1 (HIV-1) Vpu protein – a genetic, biochemical and biophysical analysis"</p><p>http://www.virologyj.com/content/4/1/81</p><p>Virology Journal 2007;4():81-81.</p><p>Published online 29 Aug 2007</p><p>PMCID:PMC2042504.</p><p></p>d white arrows indicate Vpu/ER and Vpu/Golgi colocalizations, respectively

Molecular Dynamics Simulations Reveal the HIV-1 Vpu Transmembrane Protein to Form Stable Pentamers

<div><p>The human immunodeficiency virus type I (HIV-1) Vpu protein is 81 residues long and has two cytoplasmic and one transmembrane (TM) helical domains. The TM domain oligomerizes to form a monovalent cation selective ion channel and facilitates viral release from host cells. Exactly how many TM domains oligomerize to form the pore is still not understood, with experimental studies indicating the existence of a variety of oligomerization states. In this study, molecular dynamics (MD) simulations were performed to investigate the propensity of the Vpu TM domain to exist in tetrameric, pentameric, and hexameric forms. Starting with an idealized α-helical representation of the TM domain, a thorough search for the possible orientations of the monomer units within each oligomeric form was carried out using replica-exchange MD simulations in an implicit membrane environment. Extensive simulations in a fully hydrated lipid bilayer environment on representative structures obtained from the above approach showed the pentamer to be the most stable oligomeric state, with interhelical van der Waals interactions being critical for stability of the pentamer. Atomic details of the factors responsible for stable pentamer structures are presented. The structural features of the pentamer models are consistent with existing experimental information on the ion channel activity, existence of a kink around the Ile17, and the location of tetherin binding residues. Ser23 is proposed to play an important role in ion channel activity of Vpu and possibly in virus propagation.</p> </div

Structural features of the pentamer model.

<p>(A) Kink around the Ile17 residue in the pentamer model. (B) The three residues known to interact with tetherin shown in van der Waals representation.</p