Autophagy is an intracellular biological process also known as the type II programmed cell death pathway. Autophagy is involved in numerous human diseases including cancer, muscular disorders and neurodegeneration, major histocompatability complex (MHC) antigen presentation, and innate immunity against certain bacteria and viruses [1–3]. Three types of autophagy have been well described: chaperone-mediated autophagy, microautophagy and macroautophagy. Macroautophagy represents the typical autophagic process . The hallmark of autophagy is a double-membraned autophagosome that engulfs bulk cytoplasm and cytoplasmic organelles such as mitochondria and endoplasmic reticulum . Autophagosomes ultimately fuse with lysosomes thereby generating single-membraned autophagolysosomes and degrading their contents. Autophagy is evolutionarily conserved in eukaryotes from yeast to mammals. Beclin 1, the mammalian orthologue of yeast Atg6, localizes to the trans-golgi network (TGN), mitochondria and endoplasmic reticulum as a subunit of the mammalian class III phosphatidylinositol 3-kinase (PI 3-kinase) Vps34 and mainly engages hVps34 in the autophagic pathway and in autophagosome formation [5,6]. Microtubule-associated protein (MAP) light chain 3 (LC3) is a human homologue of yeast Apg8/Aut7/Cvt5 (Atg8). MAP-LC3 is cleaved by a cysteine protease to produce LC3-I (18 kD), which is located in the cytosolic fraction. LC3-I is converted to LC3-II (16 kD) through the actions of E1- and E2-like enzymes during autophagy. LC3-II is covalently attached to phosphatidylethanolamine on its C terminus, and it binds tightly to autophagosome membranes. Therefore, LC3-II is considered the functional form of LC3 and has been used as a specific marker of autophagy [1,7–9].
Recently, Espert et al. reported that HIV proteins can induce autophagy in bystander CD4+ T lymphocytes through contact of Env with CXCR4, leading to apoptotic cell death . Based on the role of apoptosis in HIV-1 infection and the close interaction between apoptosis and autophagy, we hypothesized that HIV-1 infection may affect the autophagy pathway. Our findings indicate that the expression of Beclin 1 and the formation of autophagic vacuoles are significantly reduced during HIV-1 infection. Moreover, the reduction of Beclin 1 and autophagosome formation can be overcome by starvation of these cells.
Cell preparation and HIV-1 infection
U937 cells were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS). CD4+ T lymphocytes maintained in 50 IU/ml interleukin (IL)-2 (R&D, Minneapolis, Minnesota, USA) were purified by using a human CD4+ T Cell negative Isolation Kit II (Miltenyi Biotec, Auburn, California, USA) from healthy HIV-seronegative peripheral blood mononuclear cells (PBMC) following a 3-day treatment with 5 μg/ml phytohemagglutinin (PHA).
HIV-1MN (originally contributed by Robert Gallo through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institutes of Health, Bethesda, Maryland, USA) was propagated in H9 cells. The inactivated HIV-1MN was prepared by treating virus stocks with 1 mmol/l Aldrithiol-2 (AT-2) for 1 h at 37°C (AT-2 HIV-1)MN, as previously described [11,12].
Samples of 5 × 106 U937 or PHA-treated CD4+ T-cells in six-well plates were exposed to infectious HIV-1 at a multiplicity of infection (moi) of 5, AT-2-treated virus or supernatant from uninfected U937 cells at 37°C with 5% CO2 for 4 h. Cells were washed and incubated with RPMI 1640 with 10% FBS. HIV-1 gag p24 protein in culture supernatant of HIV-1 or inactivated AT-2 HIV-1-treated cells was measured by enzyme-linked immunosorbent assay according to the manufacturer's protocol (Beckman Coulter, Miami, Florida, USA).
To identify HIV-1 infected cells, cells were fixed with 4% paraformaldehyde (PFA). TSA Kit #2* with horseradish peroxidase-goat antimouse IgG and Alexa Fluor 488 tyramide (Invitrogen, Carlsbad, California, USA) was used according to the manufacturer's instructions. Mouse anti-HIV-1p24 monoclonal antibodies (ImmunoDiagnostics, Woburn, Massachusetts, USA) were used as the primary antibodies. Following staining, cells were examined under a fluorescent microscope.
Western blot analysis
Cytoplasmic proteins were separated on a 10 or 12% gradient Tris–glycine gel (Invitrogen) and blotted to polyvinylidine difluoride membrane. A WesternBreeze chemiluminescent western blot immunodetection kit (Invitrogen) was used according to the manufacturer's protocol. Band intensity on exposed film was semiquantified using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). Beclin 1 (BCN1), GAPDH, and MAP-LC3 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, California, USA).
Intracellular staining of LC3 protein and visualization of autophagosome
The PFA-fixed cells were blocked and permeabilized. Rabbit antihuman LC3 monoclonal antibodies and Cy3-conjugated goat antirabbit monoclonal antibodies were used for detecting autophagosomes in the cells. The stained cells was mounted on a cover-slip and analyzed on an Olympus Disk Scan Confocal Microscope system (Olympus America Inc., Center Valley, Pennsylvania, USA). The number and volume of fluorescent-stained LC3+ particles per cell were determined and used as an assessment of autophagosomes.
Autophagy is down-regulated in HIV-1-infected cells
As autophagy is known to be inhibited by serum-rich medium, autophagy formation in U937 cells and CD4+ cells was assessed in complete RPMI medium at a high density of 5 × 106 cells/ml. Using this system, Beclin1 protein in untreated cells could be detected within 2 days. The level of protein is expressed as the ratio of the Beclin 1 band to that of GAPDH. Dynamic changes of Beclin 1 protein expression were observed in U937 cells over time as the cells underwent increasing nutrient stress (Fig. 1a). With HIV-1 infection, comparable levels of autophagy were not observed until day 4 in culture, and up to day 7 the HIV-1-infected cells never achieved the levels of Beclin 1 observed in the uninfected cultures at day 2.
To examine the impact of HIV-1 infection on autophagy, U937 cells and PHA-treated CD4+ T lymphocytes were infected with HIV-1. Aldrithiol-2 (AT-2) inactivated HIV-1 (AT-2 HIV-1) was used as a control for HIV-1 infection. To ensure comparable conditions, infected and uninfected cells were treated identically at each step, including when changing culture media. Beclin 1 protein was detected by western blotting and the autographic film was analyzed and semi-quantitated with ImageJ software. The data from five independent experiments showed that the Beclin 1 expression level was significantly decreased in both U937 (P = 0.042 and CD4+ T-cells (P = 0.027) infected with HIV-1 for 48 h compared to the cells treated with media only and AT-2 treated HIV-1 (P = 0.008 for CD4+ cells and P = 0.009 for U937 cells, respectively (Fig. 1b). Beclin 1 mRNA expression levels of the cells exposed to infectious HIV-1 were also significantly decreased in comparison with that of untreated cells (data not shown). The mean reduction of mRNA expression was 28.5% in CD4+ cells (P = 0.006) and 20.5% in U937 cells (P = 0.024), respectively. The reduction of Beclin 1 caused by HIV-1 infection was confirmed further by detection of intracellular p24. In order to identify HIV-1-infected cells, a highly sensitive fluorescent signal tyramide-based staining system was employed to detect intracellular p24 . Using this detection system, approximately 80% of cells demonstrated HIV-1 infection on days 2 and 7 (Fig. 1d).
LC3 fluorescent staining was used to visualize and monitor autophagic vacuole formation during HIV-1 infection using a disk scanning confocal microscopy system that enabled analysis in three-dimensional scanning. The total number of LC3 positive (LC3+) particles and the volume of the total number of the particles in each cell were determined and used as the indicators of autophagosome formation. Following 48 h HIV-1 infection, LC3+ staining autophagocytic vacuoles were significantly reduced as measured by the volume and the number of LC3+ intracellular particles (Fig. 1e). In comparison with the uninfected cells, the mean (± SD) of LC3+ particles in infected U937 cells went from 687 ± 70 to 123 ± 74 (P < 0.001) and for CD4+ cells from 280 ± 60 to 53 ± 19 (P < 0.01), respectively. The volume of the particles observed was also significantly reduced in both cell types (P < 0.001 for U937 cells and P < 0.01 for CD4+ T cells, respectively) (data not shown). Similarly, LC3 II was markedly reduced in HIV-1-infected cells as determined by western blot (Fig. 1c).
Autophagy is induced in HIV-1-treated cells by amino acid starvation and rapamycin
During starvation, the induction of autophagy contributes to the maintenance of cellular homeostasis that helps maintain an amino acid pool for gluconeogenesis and for the synthesis of essential proteins. Starvation and rapamycin are widely used autophagic inducers that works through inhibition of mammalian target of rapamycin (mTOR), which regulates a wide array of cellular functions, including apoptosis and autophagy [14,15]. To assess whether the inhibition of Beclin 1 expression and autophagosome formation were reversible, cells were treated in serum-free Earle's balanced salts solution (EBSS) or with 200 nmol/l rapamycin at 37°C with 5% CO2 for 3 h. After induction, Beclin 1 protein was significantly increased in HIV-1-infected U937 cells (P = 0.001) and for CD4+ T lymphocytes (P = 0.044) (Fig. 2a) as were the number of autophagosomes per cell seen in CD4+ T cells (P = 0.007) and U937 cells (P = 0.012). Similarly, the volume of autophagosomes per cell was also increased in CD4+ T cells (P = 0.026) and U937 cells (P = 0.038) (Fig. 2b–e). These data demonstrate that the inhibition of autophagy by HIV-1 infection is reversible.
Our data indicate that HIV-1 infection down-regulates the expression of the autophagy protein Beclin 1 and the formation of autophagic vacuoles in U937 cells and CD4+ T lymphocytes. It is broadly reported that HIV-1 infection induces apoptosis through various viral products including envelope proteins, Vpr, Tat, Nef and protease. Among these apoptotic viral proteins, envelope protein gp120 induces apoptosis through the CD4/CXCR4–mTOR–p53 axis . In this pathway, activated mTOR, plays a central role in gp120-induced apoptosis. mTOR is a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway and is an important kinase in many cellular processes including autophagy and apoptosis. mTOR negatively controls autophagy and positively controls apoptosis. As activated mTOR inhibits autophagy, it is possible that during active HIV-1 infection envelope proteins interact with mTOR leading to a down-regulation of autophagy. Previous studies have demonstrated that gp120 activates both PKCepsilon and its upstream effector PI3K/Akt which inhibits autophagy through activating mTOR . Theoretically, factors that induce apoptosis could also inhibit autophagy due to the close relationship between autophagy and apoptosis . As amino acid starvation of the cells inhibits mTOR, the findings that starvation and rapamycin increase Beclin 1 expression and autophagic vacuole formation further support our hypothesis that HIV-1 causes the down-regulation of autophagy through mTOR activation.
As a general rule of viruses, the alterations of cellular processes induced by viral infection favors viral replication and spread. Single-stranded RNA viruses including poliovirus block the degradation of autophagosome membranes and use the membranes to anchor their RNA replication complexes [19,20]. Viruses that do not use autophagosomal membranes for their replication appear to down-regulate the formation of autophagosomes in order to enhance viral replication. HIV-1 would appear to fall into this latter category. It is likely that the down-regulation of autophagy by HIV-1 is part of an elaborate strategy used by the virus to avoid immunologic control.
In conclusion, our findings suggest HIV-1 has developed mechanisms for the down-regulation of autophagy that are probably part of a strategy designed to enhance viral replication and to evade the immune system. Additional studies of the relationship of autophagy with HIV-1 replication and survival are needed to understand the role of autophagy in HIV-1 pathogenesis.
We thank Brendan Brinkman at the UCSD Neuroscience Microscopy Shared Facility for his assistance.
Sponsorship: this study was supported in part by grants from the National Institute of Allergy and Infectious Diseases [AI-68632, AI-39004 and AI-36214 (Virology Core, University of California, San Diego, Center for AIDS Research)].
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