HIV-1 and HIV-2 infections induce autophagy in Jurkat and CD4+ T cells

Cellular Signalling 24 (2012) 1414–1419

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HIV-1 and HIV-2 infections induce autophagy in Jurkat and CD4 + T cells Xue Wang a,⁎, Yamei Gao b, Jiying Tan a, Krishnakumar Devadas a, Viswanath Ragupathy a, Kazuyo Takeda c, Jiangqin Zhao a, Indira Hewlett a,⁎ a Lab of Molecular Virology, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, United States b EM Lab, Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, United States c Confocal Microscopy Core Facility, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, United States

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Article history: Received 28 November 2011 Received in revised form 1 February 2012 Accepted 22 February 2012 Available online 2 March 2012 Keywords: HIV Autophagy Beclin-1 LC3 Electron microscopy

a b s t r a c t Autophagy plays important roles during innate and adaptive immune responses to pathogens, including virus infection. Viruses develop ways to subvert the pathway for their own benefit in order to escape restriction by autophagy, leading to increased viral replication and/or control over apoptosis of their host cells. The effects of HIV infection on the autophagic pathway in host cells have been little documented. Using the susceptible Jurkat cell line and CD4+ T cells, we studied the relationship of HIV-1 and -2 infections with autophagy. We found that HIV infections significantly increase transcription of ULK1, a member of the autophagy-initiated complex. Two ubiquitin-like conjugation systems, the Atg12 conjugation system and the microtubuleassociated protein L chain 3 (LC3) conjugation system that control the elongation of the autophore to form the autophagosome, were activated after HIV infection, with upregulation of Atg12–Atg5 complex and increased transcription of LC3, and formed more autophagosome in infected cells detected using an EM assay. We also found that HIV-1 induced more autophagic death in Jurkat cells relative to HIV-2, and the inhibition of autophagy with 3MA and Beclin-1 knockdown decreased HIV-1 replication significantly. The results indicate that HIV is able to induce the autophagic signaling pathway in HIV-infected host cells, which may be required for HIV infection-mediated apoptotic cell death. © 2012 Elsevier Inc. All rights reserved.

1. Introduction HIV-1 infection causes a progressive decline in the function and number of CD4 T lymphocytes, and high levels of viremia resulting in the development of AIDS. HIV is able to kill infected CD4expressing primary cells directly, while bystander cell killing is achieved through cells exposed to proteins associated with HIV infection [1]. Both infected and uninfected CD4 T cells have been shown to undergo cell death with apoptosis being a major death pathway to achieve cell death [2]. However, recent reports have shown that HIV-1 infection may also lead to cell death through the autophagic pathway [3–5]. Autophagy is a degradative lysosomal pathway involving the sequestration of cytoplasmic constituents (including organelles) into double-membrane-bound vesicles or autophagosomes, which eventually fuse with lysosomes for degradation. Autophagy-related (Atg) proteins modulate the formation of autophagosomes, which have been highly conserved in all eukaryotic organisms, from yeast to humans [6]. To date, 33 different autophagy-related genes have ⁎ Corresponding authors at: Laboratory of Molecular Virology, CBER/FDA, Building 29B, Rm 4NN22, 8800 Rockville Pike, Bethesda, MD 20892, United States. Tel.: + 1 301 827 0817; fax: + 1 301 480 7928. E-mail address: [email protected] (X. Wang). 0898-6568/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2012.02.016

been identified in yeast [7], and most of the corresponding homologous molecules have been confirmed in mammalian cells [8]. The Atg12 and microtubule-associated protein L chain 3 (LC3; Atg8 in yeast) conjugation systems have been shown to control the elongation of the autophore to form the autophagosome, which are known as ubiquitin-like conjugation systems. Atg7 and Atg10 catalyze Atg12 and Atg5 to form an Atg12–Atg5–Atg16L complex [9]. LC3 is cleaved by the protease Atg4 to generate the cytosolic LC3-I; Atg3 and Atg7 catalyze the conjugation of LC3-I to generate the membrane-bound lipid form, LC3-phosphatidylethanolamine, which is also called LC3-II [8]. Currently, there are only a few reports of autophagic signaling pathways including autophagic cell death in HIV infection. In 2006, it was reported that in HIV infection, envelope glycoprotein (Env) is associated with autophagy in bystander CD4 T lymphocytes [3]. HIV-1-infected cells with Env expression display autophagy and accumulation of Beclin-1, an important Atg protein, in bystander CD4 T cells independent of HIV-1 replication [3,5], which may be necessary for both apoptotic and nonapoptotic cell death required to trigger CD4 T cell apoptosis [10]. HIV-1 tat is able to induce autophagy in neuroblastoma cells [11], and autophagy is increased in postmortem brains of persons with HIV-1-associated encephalitis [12]. Alternatively, some reports demonstrated that HIV-1 infection inhibits starvation or rapamycin-induced autophagy in T cells [13] and dendritic

X. Wang et al. / Cellular Signalling 24 (2012) 1414–1419

cells [14], and in bystander macrophage/monocytic cells [15]. It has not been demonstrated whether autophagy is involved in direct killing of CD4-expressing infected cells, especially in the early stage of its infection. Here, we used a susceptible Jurkat cell line and primary CD4 T cells to study the effects of HIV infection on autophagy, and found that HIV infection increased autophagy in CD4 T cells and induced Jurkat cell death through the autophagic pathway with involvement of both Atg12 conjugation system and the microtubule-associated LC3 conjugation system. We also compared differences in autophagic pathway molecules induced by HIV-1 and HIV-2. 2. Materials and methods 2.1. Chemicals and reagents 3-Methyladenine (3MA) and other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Rabbit polyclonal antibodies against Beclin-1, Atg5 and Atg12, and siRNA (control or Beclin-1) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p62 antibody was purchased from MBL (Medical and Biological Laboratories CO (Woburn, MA)). 2.2. Cell culture The human Jurkat T cell line (clone JE6.1) was obtained from American Type Culture Collection (Manassas, VA), and cultured at 37 °C in 5% CO2 in RPMI 1640 medium containing 10% fetal calf serum, 2 mM glutamine, 50 μg/ml penicillin, and 50 μg/ml streptomycin. Cell viability was determined by trypan blue exclusion analysis (Life Technologies). CD4 T cells were isolated with CD4 T cell isolation kit (Invitrogen Carlsbad, CA), from peripheral blood mononuclear cells (PBMCs) from healthy blood donors who are seronegative for HIV-1 and HIV2, HTLV, HBV and HCV were provided by the Department of Transfusion Medicine, National Institutes of Health (NIH) (Bethesda, MD). Cells suspensions contained >95% CD4 T cells stimulated with 2 mg/ml PHA for 72 h. Activated CD4 T cells were then infected with HIV as indicated below. 2.3. HIV infection 5

Jurkat cells were seeded at 2 × 10 cells/ml for 24 h, and infected with known amounts (10 9 copies per 10 6 cells) of HIV-1 (MN) and HIV-2 (Rod) and cultured for different days indicated. CD4 T cells infected with known amounts (10 9 copies per 10 6 cells) of a primary HIV-1 subtype B virus were isolated from a US blood donor. 2.4. Electron microscopy (EM) assays Cell cultures were fixed in 2% paraformaldehyde–2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 2 h. The cells were washed with PBS buffer and then postfixed in 1% osmium tetroxide, washed with distilled water three times, dehydrated in 50%, 70%, 95% and 100% ethyl alcohol, embedded in Epon 12. Thin sections (60 nm) were cut, stained with uranyl acetate and lead citrate, and examined on a Zeiss 912 transmission electron microscope. 2.5. LC3 staining Cell cultures were washed with cold PBS twice and fixed in an acetone/methanol (vol/vol) mixture for 10 minutes at − 20 °C. Rabbit antihuman LC3 polyclonal antibodies and FITC-conjugated goat antirabbit polyclonal antibody were used for detecting autophagy in the cells, by measuring fluorescence using Zeiss Cell Observer SD Confocal Microscope system.

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2.6. Autophagy PCR array Total RNA was isolated from Jurkat cells using TRIzol® LS Reagent (InvitrogenTM, Carlsbad, CA). mRNA was isolated from total RNA using Qiagen mRNA isolation kit (Qiagen Inc., Valencia, CA). Equal amounts of mRNA per sample (0.3 μg) were reverse transcribed using RT 2 First Strand kit from SuperArray Biosciences. Comparison of the relative expression of autophagy-related genes was performed using RT 2 profiler™ PCR array PAHS-084 (human autophagy array; SABiosciences™, Frederick, MD) on a TaqMan 7500 Analyzer using RT2 Real-Time™ SYBR Green PCR master mix PA-012. Hypoxanthine phosphoribosyltransferase-1 (HPRT1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin (ACTB) “housekeeping” genes were used for normalization. 2.7. Western blot analysis Proteins were isolated from cultured Jurkat cells with RIPA buffer (1 × PBS, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0.1 mg/ml PMSF, 30 μl/ml aprotinin, and 1 mM sodium orthovanadate). For SDS–PAGE, samples containing equal amounts of protein were boiled in loading buffer (100 mM Tris–HCl, 200 mM DTT, 4% SDS, 0.2% bromphenol blue, 20% glycerol) and separated on SDS– PAGE, followed by transfer to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk and stained with primary antibodies for 2 h at the optimal concentrations. After five washes in PBS with 0.2% Tween 20, the horseradish peroxidaseconjugated secondary antibody was applied and the blot was developed with ECL reagents (Amersham Biosciences, Piscataway, NJ, USA). 2.8. siRNA transfection A small interfering RNA (siRNA) transfection kit corresponding to Beclin-1 (Human) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Jurkat cells were transfected with Beclin-1 siRNA or the control siRNA for 48 h according to the manufacturer's protocol. 2.9. Real-time PCR Quantitative real-time reverse-transcriptase (RT) PCR was used for measuring virus production. Viral RNA was isolated from 140 μl of culture supernatant by using the QIAamp Viral RNA Mini Kit (Valencia, CA 91355) according to the manufacturer's protocol. Primers and a TaqMan probe were designed in the gag p24 region of the HIV-1 subtype B isolate sequences according to the sequences in the GenBank database. The forward primer was 5′-GACATCAAGCAGCCATGCAA-3′, corresponding to nucleotides 1367–1386, and the reverse primer was 5′-CTATCC CATTCTGCAGCTTCCT-3′, corresponding to nucleotides 1430–1409. The TaqMan probe was oligonucleotide 5′-ATTGATGGTCTCTTTTAACA-3′, corresponding to nucleotides 1488–1507, coupled with a reporter dye [6-carboxy fluorescein] (FAM) at the 5′ end and a non-fluorescent quencher and a minor groove binder (MGB), which is a Tm enhancer, at the 3′ end. Nucleic acids were amplified and detected in an automated TaqMan 7500 Analyzer by using QuantiTectTM Probe RT-PCR kit (Qiagen Inc., Valencia, CA). The 25-μl PCR mixture consisted of 100 nM primers and 100 nM probe. Following three thermal steps at 55 °C for 5 min, at 50 °C for 30 min and at 95 °C for 10 min, 45 cycles of two-step PCR at 95 °C for 15 s and at 60 °C for 1 min were performed. Results were obtained from at least 3 independent experiments. 2.10. Statistical analysis The unpaired Student's t test was used for data analyses as indicated, and a value of p b 0.05 (*) was considered significant and p b 0.01 (**) very significant.

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(Fig. 2). These data indicate that both HIV-1 and HIV-2 infection are able to induce cell death directly through the autophagic pathway.

3. Results 3.1. HIV infection induces autophagosome formation

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Fundamental for cell development and survival, autophagy integrates extensively with apoptosis and may itself function as a form of programmed (type II) cell death. To examine whether HIV infection-mediated autophagy is involved in cell death, Jurkat cells, transfected with Beclin-1 siRNA, or control siRNA for 2 days, were infected with HIV-1 or HIV-2 for 3 (Fig. 2B) or 7 (Fig. 2C) days. We found that knockdown of Beclin-1 with siRNA (Fig. 2A) significantly blocked HIV-mediated cell death, relative to control siRNA treatment

The core molecular machinery driving autophagy is the Atg family of proteins, originally identified in yeast [21]. The serine/threonine kinase ULK1 is a mammalian homolog of Atg1, an upstream component of the core autophagy machinery. ULK1 has been found to be activated by glucose starvation in a manner depending on phosphorylation; AMPK (5′ adenosine monophosphate-activated protein kinase) phosphorylates ULK1 in response to cellular energy starvation to control ULK1 kinase function and autophagy induction [22,23]. Jurkat cells infected with HIV-1 or HIV-2 displayed an increased transcription of ULK1 significantly, being greater than 200 fold on day 3, and 40 fold on day 7 postinfection, relative to the uninfected control (Fig. 3A and B), suggesting that HIV infection is able to initiate autophagy through the ULK1 complex at an early infection stage. The Atg4 family of endopeptidases regulates autophagosome biogenesis by priming newly synthesized Atg8 (LC3 in mammal) to enable covalent attachment of phosphatidylethanolamine, and by delipidating LC3 at the lysosomal fusion step. One human Atg4 family member, Atg4D, is cleaved by caspase-3 during apoptosis, can be activated by H2O2 treatment and relocated to damaged mitochondria and is an important molecule acting at the regulatory interface between autophagy and apoptosis [24]. As shown in Fig. 3C and D, Jurkat cells infected with HIV-1 or HIV-2 displayed an increased transcription of Atg4D, more than 80 fold on day 7 postinfection, relative to the uninfected control. We also detected the protein expression of both ULK1 and Atg4D with Western blot analysis. As shown in Fig. 3E, HIV infection caused an increased ULK1 expression on day 3 postinfection and more Atg4D

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3.2. Autophagy is involved in HIV infection-mediated cell death

3.3. Increased transcription and expression of ULK1 and Atg4D after HIV infection

% of cells with autophagosome

Autophagy was first described by electron microscopy (EM) approximately 50 years ago [16,17] and EM remains one of the most widely used and sensitive techniques to detect the presence of autophagic vesicles [18]. Autophagy induced by cell signaling starts when a flat membrane cistern wraps around a portion of cytoplasm and forms a closed double-membrane vacuole containing cytosol and/or organelles [19]. The sealed vacuole is called the autophagosome, an early autophagic compartment. Autophagosomes mature by fusing with endosomal and lysosomal vesicles, which also deliver lysosomal membrane proteins and enzymes [20]. To examine whether HIV infection induces autophagy that is able to be represented by autophagosome, Jurkat cells were infected with HIV-1 or HIV-2 for 7 days and analyzed with electron microscopy. As shown in Fig. 1, HIV infection increased the formation of autophagosome significantly compared with the uninfected control, suggesting that both HIV-1 and HIV-2 infection induce autophagy directly in infected Jurkat cells. HIV-1 induced more autophagosome formation relative to HIV-2 infection (Fig. 1D).

Fig. 1. HIV infection induced more autophagosome formation. Jurkat cells were infected with HIV-1 (MN) (B) and HIV-2 (Rod) (C) for 7 days, and cells without infection were used as control (A), and then cell pellets were subjected to fix, stain, and assay with electron microscope. Arrows represented autophagosome; bar represented 1 μm. (D) HIV infection induced more autophagosome formation in the cells; the data are presented as mean percent autophagosome following evaluation of about 8 cells/field in randomly selected at least 10 fields.

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Fig. 2. HIV infections induced autophagic cell death. Jurkat cells, transfected with control siRNA or Beclin-1 siRNA for 2 days, were infected with HIV-1 or HIV-2 for another 3 (B) or 7 (C) days indicated. Cell viability was subjected to trypan blue exclusion analysis to detect the effects of HIV infections on cell death relative to Jurkat cell transfected with control siRNA without HIV infection. Cell lysates were subjected to Western blot analysis to detect Beclin-1 (A).

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expression in day 7 postinfection, suggesting that Jurkat cells infected with HIV displayed more ULK1 expression early and more Atg4D expression late.

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Fig. 3. HIV infection increased autophagic gene, UKL1 and Atg4D, expression. Jurkat cells were infected with HIV-1 or HIV-2 for 3 days or 7 days indicated. mRNA was isolated and cDNA array was performed with PCR array to detect the transcription of UKL1 on day 3 (A) or day 7 (B) postinfection and Atg4D on day 3 (C) or day 7 (D) postinfection relative to a uninfected control. Cell lysates were subjected to Western blot analysis to detect UKL1 and Atg4D (E).

While infection with HIV-1 results in immunodeficiency and death in the majority of untreated patients, approximately 80% of those infected with HIV-2 tend to remain long-term nonprogressors, having a normal life expectancy, low or undetectable plasma virus load and an intact immune system with high CD4 lymphocyte counts [2]. To examine the potential differences in effects of HIV-1 and HIV-2 on the autophagic pathway, Jurkat cells were infected with HIV-1 or HIV-2 for 3 days or 7 days as indicated. As shown in Fig. 4A, HIV-1 infection caused more Atg4D transcription relative to infection with HIV-2 on day 3 postinfection, and more autophagosome formation (Fig. 1D) and higher degree of cell death (Fig. 2) were induced by HIV-1 relative to HIV-2 infection. Beclin-1 (Atg6 in yeast), first described as a Bcl-2-interacting protein, forms a complex with human VPS34, class III phosphatidylinositol 3-kinase, to control human VPS34-mediated vesicle trafficking pathways including autophagy. Beclin-1 is required for Atg5/Atg7dependent and -independent autophagy [25]. HIV-1 infected Jurkat cells displayed higher expression of Becin-1 at both levels of mRNA (Fig. 4A) and protein (Fig. 4C). Atg3 interacts with Atg12, which it is necessary for the formation of Atg12–Atg5 conjugates and for the elongation and closure of autophagosomes [24]. HIV-1 infection causes more Atg3 and Atg4C transcription relative to HIV-2 infection in Jurkat cells (Fig. 4B). The p62 protein has been identified as one of the specific substrates that are degraded through the autophagy–lysosomal pathway [26]. This degradation is mediated by interaction with LC3, which is recruited to the phagophore/isolation membrane and remains associated with the completed autophagosome [27]. HIV-1 infection was shown to cause high expression of LC3 (Fig. 4B) and p62 (Fig. 4C) relative to HIV-2 in Jurkat cells. We also tested the effect of HIV infection on the formation of Atg12–Atg5 conjugates, and found that Jurkat cells infected with HIV displayed formation of Atg12–Atg5 conjugate relative to the

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uninfected control (Fig. 4C). Additionally, greater levels of the complex of Atg12–Atg5 were formed in Jurkat cells infected with HIV-1 than HIV-2 (Fig. 4C). These data indicate that HIV infection directly induces cell death through the autophagic pathway. HIV-1 induces more autophagic cell death, with a higher degree of formation of Atg12–Atg5 conjugate, and increased expression of Atg3, Atg4, Beclin-1, LC3, and p62. 3.5. HIV-1 infection induces autophagy with Beclin-1 and LC3 expression in CD4 + T cells To verify whether HIV-1 infection causes autophagy in primary cells, CD4+ T-cells were isolated and infected with HIV-1 clade B (#9697) for 3 days. HIV-1 infection significantly increased expression of Beclin-1 (Fig. 5A) and LC3 (Fig. 5B), suggesting that HIV infection is able to induce autophagy in a T cell line and primary CD4+ T cells. 3.6. Autophagy inhibition decreases HIV-1 virus production A recent report showed that inhibition of autophagy decreases HIV1 virus production in macrophages [27]. To test whether autophagy inhibition decreases HIV-1 replication, Jurkat cells were infected with HIV-1 for 7 days, and then treated with 3-methyl adenine (3MA), a conventional inhibitor of autophagy, for an additional 2 days; viral RNA was isolated from cell-culture supernatants. As shown in Fig. 6A, 3MA treatment decreased virus production significantly relative to control. To test the effect of Beclin-1 on HIV-1 virus production, Jurkat cells, transfected with small interfering RNA of Beclin-1 for 2 days, were infected with HIV-1 for 7 days, and viral RNA was measured in cell-culture supernatants. Transfection with Becin-1 siRNA caused a significant decrease in HIV-1 virus production relative to control siRNA (Fig. 6B). 4. Discussion HIV infection is known to cause progressive CD4 T cell depletion and induces death through direct killing of host cells or bystander

Fig. 5. HIV-1 infection induces autophagy in CD4+ T cells. CD4 T cells infected with HIV-1 subtype B isolate (#9897) for 3 days. Cell lysates were subjected to Western blot analysis to detect Beclin-1(A); some CD4 T cell pellets were subjected to fix, stain, and assay with Zeiss Cell Observer SD Confocal Microscope system to detect LC3 (B), bar represented 20 μm.

killing by proteins from HIV particles. It has been well accepted that apoptosis of uninfected bystander CD4 T lymphocytes plays a major role in AIDS development [1]. We have previously shown that both HIV infection and HIV-1 envelope glycoprotein (Env), gp120 treatment, can induce Jurkat cell death through apoptotic pathways [2,28]. Treatment with HIV-1 Env has been showed to induce autophagic death of CD4 T lymphocytes, and this autophagy process is required to trigger CD4 T cell apoptosis since inhibition of autophagy at different steps, by either drugs or siRNAs specific for autophagic genes, totally abolished apoptosis [3–5]. This may indicate that HIV1-infected cells can induce death of bystander CD4 T lymphocytes

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Fig. 4. HIV infection increased more expression of autophagic genes. Jurkat cells were infected with HIV-1 or HIV-2 for 3 days or 7 days indicated, and mRNA isolated was subjected to cDNA array to detect the transcription of Atg4D and Beclin-1 (A), Atg3, Atg4C and LC3 (B) on day 3 postinfection. Cell lysates were subjected to Western blot analysis to detect Beclin-1, p62 and complex of Atg12–Atg5 (C).

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Fig. 6. Autophagic inhibition decreased HIV-1 virus production. Jurkat cells were infected with HIV-1 (MN) for 7 days, and then washed and incubated with control media or 3MA for another 2 days. 140 μl of culture supernatants containing HIV-1 particles were used to isolate viral RNA. 10 μl in 50 μl of the RNA was used as template to perform real-time PCR (A). Jurkat cells, transfected with siRNA Beclin-1 or siRNA control for 2 days, were infected with HIV-1 (MN) for another 7 days. 140 μl of culture supernatants containing HIV-1 particles were used to isolate viral RNA. 10 μl in 50 μl of the RNA was used as template to perform real-time PCR (B). Known concentrations of HIV-1 (MN) viral RNA (serially diluted: 108 to 100 copies) were used as templates and quantitative RT-PCR performed to generate a standard curve. Each value represents the average concentration of six reactions in triple isolated repeats based on the standard curve.

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through contact with Env and trigger cell death pathways in a sequential order of autophagy followed by apoptosis [10]. Autophagy mediates the lysosome-dependent turnover of macromolecules and entire organelles, induced by multiple forms of cellular stress, including nutrient or growth factor deprivation, hypoxia, reactive oxygen species, DNA damage, protein aggregates, damaged organelles, or intracellular pathogens, and cytosolic components are delivered to the vacuole (the lysosome analog in yeast) through the autophagy pathway and are degraded by resident hydrolases [6–8]. The mammalian target of rapamycin (mTOR) kinase, a major controller of cap-dependent translation in cells, strongly inhibits autophagy. When nutrients are replete, mTOR associates with ULK1–Atg13–FIP200 complexes on autophagic isolation membranes and hyper-phosphorylates ULK1 and Atg13 which suppresses isolation membrane expansion [6]. Nutrient deprivation or the drug rapamycin inhibit the association of mTOR and ULK1 and/or mTOR kinase activity which keeps ULK1 and Atg13 hypophosphorylated and leads to the upregulation of autophagy in both mammalian cells and yeast [6–8]. HIV infection increases ULK1 expression significantly, especially at earlier time (on day 3 postinfection) of infection (Fig. 3A and B). Two ubiquitin-like conjugation systems, the Atg12 conjugation system and the microtubule-associated protein L chain 3 (LC3) conjugation system, control the elongation of the autophore to form the autophagosome. In the first system, an Atg16–Atg12–Atg5 heterotrimeric complex is formed, which associates primarily on the outer membrane of growing autophagosomes where it is hypothesized to mediate curvature of the growing membrane [8,24]. The second ubiquitin-like conjugation system results in the cleavage of LC3 by Atg7 and the protease Atg4. After cleavage, the E2-like enzyme Atg3 adds phosphatidylethanolamine (PE) to a conserved glycine residue in the C terminus of the cleaved LC3 to create a species known as LC3-II or LC3-PE [8,27]. HIV infections induce Jurkat cell autophagic death, and activate both ubiquitin-like conjugation systems with an increased LC3 transcription (Fig. 5B) and formation of Atg12–Atg5 complex (Fig. 5C) to induce formation of the autophagosome (Fig. 1). HIV-1 infection has been reported to inhibit starvation or rapamycin-induced autophagy in CD4+ T cells (13), dendritic cells [14], or macrophage/monocytic cells (15). These findings are consistent with the results reported here using CD4+ T cells or Jurkat cells with downregulation of Beclin-1 and LC3 (Sup. 1). These findings suggest that HIV-1 infection decreases autophagy induced by serumstarvation (Sup. 1B) and may result from modulation of Akt and Stat3 expression by HIV (15) (Sup. 2). These effects need to be studied further in order to more fully understand the role of autophagy in HIV infection. Many molecules can regulate autophagy, such as JNK, ERK1/2, AMP kinase, class I and class III PI3K, Akt, mTOR, JAK, STAT, eIF2α kinases, DAPK, Bcl-2 family proteins, the p53 tumor suppressor, and FLIP [29]. We have previously shown that HIV-1 infection increases JNK expression and decreases ERK1/2 expression relative to HIV-2 infection [2]. HIV-1 induces a higher degree of cell death through stronger activation of both apoptotic pathways. HIV-1 infection downregulates both Bcl-XL and FLIP expressions at later time points postinfection, while HIV-2 infection dramatically upregulated both Bcl-XL and FLIP expression [2]. The difference in cell autophagic death between HIV-1 and HIV-2 infection may be due to different effects on the expression of the autophagy regulatory molecules, such as JNK, ERK1/2, Bcl-XL, or FLIP, and need further investigation.

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