Abstract
Autophagy is a self-digestion pathway essential for maintaining cellular homeostasis and cell survival and for degrading intracellular pathogens. Human immunodeficiency virus-1 (HIV-1) may utilize autophagy for replication as the autophagy-related protein-7 (ATG-7), microtubule-associated protein 1 light chain 3, ATG-12, and ATG-16L2 are required for productive HIV-1 infection; however, the effects of autophagy induction on HIV-1 infection are unknown. HIV-1-infected individuals have lower levels of 1α,25-dihydroxycholecalciferol, the hormonally active form of vitamin D, than uninfected individuals. with the lowest concentrations found in persons with AIDS. Using human macrophages and RNA interference for ATG-5 and Beclin-1 and chemical inhibition of phosphatidylinositol 3-kinase, we have found that physiologically relevant concentrations of 1α,25-dihydroxycholecalciferol induce autophagy in human macrophages through a phosphatidylinositol 3-kinase-, ATG-5-, and Beclin-1-dependent mechanism that significantly inhibits HIV-1 replication in a dose-dependent manner. We also show that the inhibition of basal autophagy inhibits HIV-1 replication. Furthermore, although 1α,25-dihydroxycholecalciferol induces the secretion of human cathelicidin, at the concentrations produced in vitro, cathelicidin does not trigger autophagy. Our findings support an important role for autophagy during HIV-1 infection and provide new insights into novel approaches to prevent and treat HIV-1 infection and related opportunistic infections.
Highlights
One-third of Human immunodeficiency virus-1 (HIV-1)-infected individuals are co-infected with Mycobacterium tuberculosis, a leading cause of death among people living with HIV-1
Immunoblotting—Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (14C10), LC3B (D11), PI3KC3 (D9A5), Beclin-1, and ATG5 antibodies were obtained from Cell Signaling, HIV-1 Nef (3D12) antibody was from Abcam, and -actin antibody (AC-74) was from Sigma
As autophagy proteins are required for efficient HIV-1 replication in HeLa cells [16], to determine whether 1,25D3 affects HIV-1 replication in macrophages through autophagy, we examined whether the inhibition of sequential steps of the autophagy pathway inhibits HIV-1 replication
Summary
Cells and Reagents—Monocyte-derived macrophages were obtained by culturing monocytes isolated from the peripheral blood mononuclear cells of HIV-1 seronegative donors (with approval from the University of California San Diego Institutional Review Board) in RPMI 1640 (Invitrogen) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gemini Bio-Products) and 10 ng/ml macrophage colony stimulating factor (R&D Systems) for 10 days at 37 °C, 5% CO2. Cells were washed extensively with Dulbecco’s phosphate-buffered saline at 4 °C to remove unbound virions and lysed in 120 l of CelLytic M (Sigma) supplemented with protease inhibitors (Thermo Scientific). Immunoblotting—Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (14C10), LC3B (D11), PI3KC3 (D9A5), Beclin-1, and ATG5 antibodies were obtained from Cell Signaling, HIV-1 Nef (3D12) antibody was from Abcam, and -actin antibody (AC-74) was from Sigma. Fluorescence Microscopy—Cells were fixed and permeabilized in Dulbecco’s phosphate-buffered saline supplemented with 4.5% (w/v) paraformaldehyde and 0.1% (v/v) saponin for 30 min, washed, probed with mouse anti-Gag-p17 (2D11) (Abcam) and rabbit anti-LC3B (D11) for 30 min. Intracellular staining of endogenous saponin-resistant LC3B was performed as previously described [30] using rabbit anti-LC3B (D11) and allophycocyanin-conjugated mouse anti-rabbit antibodies (BD Biosciences). Statistics—Unpaired, two-tailed, Student’s t tests, ␣ ϭ 0.05 were used to assess whether the means of two normally distributed groups differed significantly
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