Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Feb;77(4):2539-49.
doi: 10.1128/jvi.77.4.2539-2549.2003.

Phosphatidylinositol 3-kinase regulates human immunodeficiency virus type 1 replication following viral entry in primary CD4+ T lymphocytes and macrophages

Fleur François  1 , Mary E Klotman
Affiliations

Phosphatidylinositol 3-kinase regulates human immunodeficiency virus type 1 replication following viral entry in primary CD4+ T lymphocytes and macrophages

Fleur François et al. J Virol. 2003 Feb.

Abstract

Human immunodeficiency virus (HIV) gp120 induces multiple cellular signaling pathways, including the phosphatidylinositol 3-kinase (PI3-kinase) pathway. The role of the PI3-kinase pathway in HIV-1 replication is not understood. Here we examined whether HIV-1 gp120 upregulates the PI3-kinase pathway and whether PI3-kinase activity plays a role in virus replication in primary human CD4(+) T cells and macrophages. Soluble and virion-associated HIV-1 gp120 induced calcium mobilization and phosphorylation of the PI3-kinase downstream effectors PKB/Akt and p70 S6 kinase. gp120-induced PI3-kinase activity and calcium mobilization were inhibited by pertussis toxin and blocking antibodies directed against CCR5 and CXCR4, suggesting that the signaling is mediated through the chemokine receptor. The PI3-kinase inhibitor LY294002 inhibited infection of CD4(+) T cells and macrophages with X4 and R5 HIV-1-pseudotyped viruses at concentrations that did not induce cell toxicity or downregulate HIV-1 coreceptor expression. When gp120-induced signaling was bypassed with the vesicular stomatitis virus G envelope protein, infection was still sensitive to PI3-kinase inhibition, suggesting that basal PI3-kinase activity is required for infection. LY294002 inhibited HIV-1 infection when added after viral entry and did not affect formation of the HIV-1 reverse transcriptase products R/U5 and long terminal repeat/Gag in the presence of the inhibitor. However, when the inhibitor was added after viral integration had occurred, no inhibition of HIV infection was observed. Our studies show that inhibition of the PI3-kinase signaling pathway suppresses virus infection post-viral entry and post-reverse transcription but prior to HIV gene expression. This type of host-virus interaction has implications for anti-HIV therapeutics that target cellular signaling machinery.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
HIV-1 gp120 induction of calcium mobilization in primary CD4+ T lymphocytes. (A) Phytohemagglutinin-activated CD4+ T lymphocytes were loaded with the calcium indicator dye indo-1 AM (20 μM), and the ratio of FL5/FL6 fluorescence was analyzed by flow cytometry immediately following treatment with 10 μM ionomycin and HBSS as positive and negative controls, respectively (left panel), 20 nM oligomeric IIIB (X4) g120 (middle panel), or 20 nM BaL (R5) gp120 (right panel). Acquisition data shown are representative of four independentexperiments. (B) Data acquired in A were analyzed with the FlowJo kinetics platform. The percentage of cells responding to gp120 in A is shown. (C) Calcium mobilization in activated CD4+ T cells in response to either control mock virus preparation lacking envelope, 15 ng of reverse transcriptase of JRFL (R5) HIV-1-pseudotyped virus per ml, or 50 nM MIP-1α. Calcium flux was measured as described above (A and B), with percentages of cells responding to each stimulation shown. To demonstrate specificity, T cells were treated with anti-CCR5 (gray bars) or mouse isotype control IgG2b (black bars) antibodies for 15 min at 37°C before stimulation. The anti-CCR5 antibody completely blocked calcium flux in response to JRFL. Data are representative of two independent experiments.
FIG. 2.
FIG. 2.
HIV-1 gp120 induction of PI3-kinase activity in CD4+ T lymphocytes. (A) Phytohemagglutinin-activated CD4+ T lymphocytes were treated with 10 nM oligomeric IIIB (X4) gp120 for the times indicated at 37°C. Total protein was extracted and analyzed by immunoblotting the same blot with antibodies specific for the proteins indicated at the right of the panels. Data are representative of five independent experiments. (B) Activated CD4+ T cells were treated with 10 nM BaL (R5) gp120 for the times indicated at 37°C. Total protein was extracted and analyzed as in A. Data are representative of three independent experiments. (C) Activated CD4+ T cells were treated with anti-CXCR4 antibody (+) or mouse IgG2b isotype control (−) for 30 min at 37°C prior to treatment with 15 ng of reverse transcriptase of concentrated HXB2 (X4) HIV-1-pseudotyped luciferase virus per ml for 5 and 15 min at 37°C. (D) Phosphorylation of p70 S6 kinase in C was analyzed by immunoblotting, and densitometry results are expressed as the increase in the levels of phosphorylated protein over that in the untreated control. Results are shown for cells treated with anti-CXCR4 antibody (gray bars) or IgG2b isotype control (black bars). Data are representative of two independent experiments.
FIG. 3.
FIG. 3.
Effect of LY294002 on HIV-1 infection of CD4+ T lymphocytes. (A) Activated CD4+ T cells were treated with 20 μM LY294002 (gray bars) or DMSO control (black bars) for 1 h at 37°C prior to infection with either HXB2 (X4) or JRFL (R5) HIV-1-pseudotyped luciferase virus. Luciferase activity was measured 24 h postinfection and normalized to total protein concentration. Uninfected background levels of luciferase were subtracted from total luciferase activity. Results are expressed as a percentage of the DMSO control infection. Data are averages from four or more independent experiments, where * and ** indicate P < 0.0001. Inset, confirmation of PI3-kinase inhibition by immunoblotting for phosphorylated Akt (a) and total Akt protein (b) following LY294002 treatment. (B) Activated CD4+ T cells were treated with DMSO (gray bars) or 20 μM LY294002 (black bars) for 60 min at 37°C prior to staining with allophycyanin-, fluorescein isothiocyanate-, and phycoerythrin-conjugated antibodies against CD4, CCR5, and CXCR4 and their corresponding isotype controls. Fluorescence staining was measured by flow cytometry, and data are averages of two independent experiments, with the ranges indicated. (C) Cell viability of activated CD4+ T lymphocytes incubated with 20 μM LY294002 or DMSO control for 24 h at 37°C measured with the Promega CellTiter96 aqueous MTS assay. Over 90% of cells were viable after 24 h of incubation with 20 μM LY294002 (P > 0.05). (D) Activated CD4+ T cells were treated with 20 μM LY294002 in the absence or presence of 50 μM z-VAD-FMK for 1 h at 37°C prior to infection with HXB2-HIV-1-pseudotyped luciferase virus. Percenta infection was determined as described for A. Data are averages of two independent experiments, with the range indicated by bars.
FIG. 4.
FIG. 4.
Effect of LY294002 on HIV-1 infection of macrophages. (A) Primary human macrophages were treated with 5 to 20 μM LY294002 or the DMSO control for 1 h at 37°C prior to infection with JRFL-HIV-1-pseudotyped luciferase virus. Twenty-four hours postinfection, luciferase activity and total protein concentrations were measured. Infection is expressed as relative luciferase units (R.L.U.), normalized to total protein concentration minus background. Infected samples (black bars) and uninfected control samples (gray bar) are indicated. Data are representative of two independent experiments. Error bars represent standard deviations of triplicate samples. (B) Macrophages were treated with 20 μM LY294002 or DMSO control for 1 h at 37°C prior to infection with JRFL-HIV-1-pseudotyped luciferase virus. Twenty-four hours postinfection, luciferase activity was measured as in A. Results are expressed as percentage of infection of the DMSO-treated control. Data are averages from three or more independent experiments (*, P < 0.0001). (C) Macrophages were treated with 20 μM LY294002 in the absence or presence of 50 μM z-VAD-FMK for 1 h at 37°C prior to infection with JRFL-HIV-1-pseudotyped luciferase virus. Percent infection was determined as in B. Data are averages of three independent experiments.
FIG. 5.
FIG. 5.
LY294002 inhibits HIV-1 infection of CD4+ T cells post-viral entry. Activated CD4+ T cells were treated either for 1 h before infection or immediately after virus entry with DMSO, 20 μM LY294002, 100 nM TAK779, 1 μM AMD3100, or 1 mM zidovudine. Twenty-four hours postinfection, cells were harvested, and percent infection was determined as described for Fig. 3. Results are averages of two or more independent experiments for infection with HIV-1 luciferase viruses pseudotyped with JRFL (A), HXB2 (B), or vesicular stomatitis virus (C). For vesicular stomatitis virus (C), LY294002 was also added 24 h postinfection, and cells were harvested after an additional 24 h of incubation.
FIG. 6.
FIG. 6.
LY294002 inhibits HIV-1 infection of CD4+ T cells post-viral reverse transcription. (A) Semiquantitative PCR was performed on DNA extracted from CD4+ T cells 24 h postinfection with HXB2- and JRFL-pseudotyped HIV-1 luciferase viruses following treatment with DMSO or 20 μM LY294002. Primers were used to amplify the HIV-1 reverse transcriptase products R/U5 (top panel) and long terminal repeat (LTR)/Gag (middle panel) as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (bottom panel) as a control. In parallel, PCR was performed on an HIV-1 sequence-containing plasmid with defined copy number to construct a standard curve. Data are representative of three independent experiments. (B) Quantification of data shown in panel A was performed with the Alpha Innotech FluorChem 8800 imaging system. Copy numbers of HIV-1 R/U5 (topgraph) and HIV-1 long terminal repeat/Gag (bottom graph) are shown for LY294002-treated samples (gray bars) and DMSO controls (black bars). There was no significant difference in long terminal repeat/Gag copies between LY294002- and DMSO-treated infected cells (P > 0.5) and that in R/U5 copies between LY294002- and DMSO-treated infected cells (P > 0.1).
See this image and copyright information in PMC

References

    1. Adachi, A., H. E. Gendelman, S. Koenig, T. Folks, R. Willey, A. Rabson, and M. A. Martin. 1986. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol. 59:284-291. - PMC - PubMed
    1. Alfano, M., H. Schmidtmayeriva, C. A. Amella, T. Pushkarsky, and M. Bukrinsky. 1999. The B-oligomer of pertussis toxin deactivates CC chemokine receptor 5 and blocks entry of M-tropic HIV-1 strains. J. Exp. Med. 190:597-605. - PMC - PubMed
    1. Alkhatib, G., M. Locati, P. E. Kennedy, P. Murphy, and E. A. Berger. 1997. HIV-1 coreceptor activity of CCR5 and its inhibition by chemokines: independence from G protein signaling and importance of coreceptor downmodulation. Virology 234:340-348. - PubMed
    1. Arthos, J., A. Rubbert, R. L. Rabin, et al. 2000. CCR5 signal transduction in macrophages by human immunodeficiency virus and simian immunodeficiency virus envelopes. J. Virol. 74:6418-6424. - PMC - PubMed
    1. Baba, M., O. Nishimura, N. Kanzaki, et al. 1999. A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. Proc. Natl. Acad. Sci. USA 96:5698-5703. - PMC - PubMed

Publication types

Substances

LinkOut - more resources