Drug 9AA reactivates p21/Waf1 and Inhibits HIV-1 progeny formation
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It has been demonstrated that the p53 pathway plays an important role in HIV-1 infection. Previous work from our lab has established a model demonstrating how p53 could become inactivated in HIV-1 infected cells through binding to Tat. Subsequently, p53 was inactivated and lost its ability to transactivate its downstream target gene p21/waf1. P21/waf1 is a well-known cdk inhibitor (CKI) that can lead to cell cycle arrest upon DNA damage. Most recently, the p21/waf1 function was further investigated as a molecular barrier for HIV-1 infection of stem cells. Therefore, we reason that the restoration of the p53 and p21/waf1 pathways could be a possible theraputical arsenal for combating HIV-1 infection. In this current study, we show that a small chemical molecule, 9-aminoacridine (9AA) at low concentrations, could efficiently reactivate p53 pathway and thereby restoring the p21/waf1 function. Further, we show that the 9AA could significantly inhibit virus replication in activated PBMCs, likely through a mechanism of inhibiting the viral replication machinery. A mechanism study reveals that the phosphorylated p53ser15 may be dissociated from binding to HIV-1 Tat protein, thereby activating the p21/waf1 gene. Finally, we also show that the 9AA-activated p21/waf1 is recruited to HIV-1 preintegration complex, through a mechanism yet to be elucidated.
KeywordsKinase Assay Uninfected Cell ACH2 Cell Small Chemical Molecule Preintegration Complex
Amid the availability of current diverse classes of anti-HIV reagents such as reverse transcriptase (RT), protease (PR) and fusion inhibitors, the development of inhibitors targeting another important HIV enzyme IN (integrase) has stimulated great interests in that there is an absence of cellular homologues of IN in cells. A number of HIV-1 IN inhibitors have been identified and few have been clinically examined including GS-9137 [1, 2, 3] and MK-0518 . Recently, increasing evidence has indicated that small chemical molecules, such as nucleoside antiretroviral reagents, may be advantageous over other antiviral reagents, since they have long intracellular half-life and low protein binding properties [5, 6, 7]. So far few detailed studies have been carried out in investigating the role of these small chemical molecules involved in the signaling pathway(s) of HIV-infected cells. However, exploring whether there are novel small chemical molecules or small peptides that can reactivate important cell signaling pathways (i.e., p53 and/or p21/waf1) that are normally inactivated in HIV-1 infected cells may help to identify important mechanisms that play key roles in HIV-1 infection and provide a new set of preventive and/or therapeutic drug-targets.
P53 pathway has been revealed to play an important role in HIV-1 infection [8, 9]. It was also shown that p53 is involved in apoptosis, where cell death can be induced by HIV-1 envelope through mTOR-mediated phosphorylation of p53 on Ser15 [10, 11]. Our previous work established a model where p53 was inactivated in HIV-1 infected cells through binding to Tat protein. Subsequently, p53 was inactivated and lost its ability to transactivate its downstream target gene p21/waf1 . The interplay between p53 and HIV-1 Tat has also been extensively studied. An RGD-containing domain of Tat protein, Tat-(65–80), was found to be important in regulating the proliferative functions of a variety of cell lines, including a human adenocarcinoma cell line, A549. The p53 activity was greatly reduced when cells were treated with Tat-(65–80) . Tat was also shown to efficiently inhibit the transcription of p53 both in vivo and in vitro. The downregulation of p53 by Tat may be important in the establishment of productive viral infection in cells and also may be involved in the development of AIDS-related malignancies .
The regulation of the p53 and p21/waf1 pathways by HIV-1 infection has become a point of interest. Previous studies have shown that the effects of p21/waf1 are highly cell-type specific in HIV-1 infection. In macrophages, HIV-1 infection resulted in an increased expression of p21/waf1 . Repression of HIV-1 replication was observed when p21/waf1 expression was inhibited by small molecules like compound CDDO (2-Cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid) [16, 17]. This phenomenon was different from that of p21/waf1 in HIV-1 infection in T lymphocytes, where HIV-1 infection reduced the expression of p21/waf1 . It also differs in stem cells, where silencing p21/waf1 expression by siRNA increased the viral replication . Therefore, we reasoned that it is possible to inhibit the HIV-1 infection and viral replication through the restoration of p53 and p21/waf1 pathways using small chemical molecules or small peptides. More recently, the p21/waf1 function was investigated as a molecular barrier for HIV-1 infection in stem cells . Hematopoietic stem cells were previously demonstrated to be highly resistant to HIV-1 infection [19, 20, 21]. In the study carried by Zhang J et al, the cdk inhibitor p21/waf1 was revealed to restrict HIV-1 infection in primitive hematopoietic cells. By knockdown of the endogenous p21/waf1 level using siRNAs, the stem cells became highly susceptible to HIV-1 infection. Further, it was shown that the effect of p21/waf1 is specific; silencing other p21/waf1 related proteins, p27 and p18 had no effect on HIV-1 infection. Finally, the authors demonstrated a novel mechanism in which the anti-HIV effect of p21/waf1 was the result of its interaction with HIV-1 preintegration complex (PIC). Therefore, p21/waf1 was suggested to be a possible restriction factor, similar in function to the TRIM5 and APOBEC3G genes [22, 23, 24, 25, 26]. Similarly, our previous work has shown that high-titer infection of HIV-1 in T lymphocytes resulted in a loss of the endogenous p21/waf1 , further demonstrating the importance of p21/waf1 in HIV-1 biology.
In this report, we show that a small chemical molecule 9AA can efficiently reactivate the p53 pathway in HIV-1 infected cells, and accordingly transactivates its downstream gene p21/waf1, a gene that has a potential role in inhibiting viral replication. We have also observed that the effect of 9AA on HIV-1 viral replication and virus DNA integration in HIV-1 infected cells is due to the association of 9AA-induced p21/waf1 with HIV-1 preintegration complex (PIC). The implication of these findings will be discussed below.
Drug 9AA reactivates the p53 and p21/waf1 pathways in HIV-1 infected cells
We have previously reported that in HIV-1 infected T-cells, p53 was inactivated through binding to HIV-1 Tat protein and the expression of p21/waf1 was nearly completely inhibited as a consequence of the inactivation of p53 . Small molecules, such as leptomycin B, actinomycin D, and 9AA (9-aminoacridine), were demonstrated to be able to efficiently reactivate p53 in some cancer cell lines [27, 28, 29]. Therefore, we reasoned that restoration of the p53 function may provide a new way to combat virus infection where this pathway is normally impaired or sequestered [30, 31, 32]. In this study, we specifically used 9AA and tested whether it could efficiently restore the functions of p53 and p21/waf1 in HIV-1 infected cells.
Effect of increased p21/waf1 in infected and uninfected cells
Effect of 9AA in PBMC infected cells
Effect of phosphorylation of serine 15 p53 on Tat binding
Drug 9AA induces p21/waf1 and its recruitment into pre-integration (PIC) complex
9AA-treatment involved in post-reverse transcriptional processes of HIV-1 infection
Viral mRNA levels present in CEMs under different treatments
mRNA level (24 hrs)
mRNA level (48 hrs)
mRNA level (72 hrs)
P53 was previously shown to be inactivated by HIV-1 infection in T-cells, and consequently downregulates the expression of its target gene p21/waf1 . In this study, we demonstrated that the function of p53 and p21/waf1 pathways could be restored by using a small chemical molecule 9-aminoacridine (9AA) (Figure 1). Very interestingly, 9AA was shown to differentially trigger the activation of p53 in HIV-1 infected and uninfected cells (Figure 1). P53 is present at low levels under unperturbed conditions, but it becomes rapidly activated and stabilized upon induction by a number of stimuli, including the use of reagents that cause DNA damage [40, 41, 42, 43, 44, 45]. Phosphorylation plays a critical role in the activation and stabilization of p53. Of particular interest is the phosphorylation of ser15, which is generally considered to be activated in response to different stress signals [46, 47, 48, 49, 50]. The p53 pathway has been demonstrated to play a key role in HIV-1 infection [8, 9]. Previous work in our lab has established a model demonstrating how p53 could become inactivated in HIV-1 infected cells through binding to Tat. P53 was inactivated and lost its ability to transactivate its downstream target gene p21/waf1 . In our current study, we show that the 9AA-triggered phosphorylated p53ser15 does not interact with HIV-1 Tat protein. One possible explanation may be that the p53ser15 is located in the core pocket domain which is required for the p53-Tat interaction, while the phosphorylation of ser15 greatly reduces the binding affinity to Tat protein.
In the current study we propose that it is feasible to reduce the HIV-1 infection and viral replication through the restoration of p53 and p21/waf1 pathways by using small chemical molecules or small peptides. When HIV-1 PBMCs were treated with 9AA at a concentration-dependent manner (0, 0.01, 0.5, 1.0 uM), the viral replication was significantly inhibited at 0.5 uM (Figure 3A), while the cell growth was not greatly affected (Figure 3B). Further, we performed an in vitro kinase assay with another HIV-1 positive cell line ACH2 treated with 9AA. We immunoprecipited cdk2/cycle E complex from the drug treated and untreated samples and the results show that 9AA induced an inhibition of the kinase activity of cdk2/cycle E complex (Figure 2), indicating that the HIV-1 infected cell line(s) may be more sensitive to the drug treatment, as compared to the HIV-1 negative cell line(s).
P21/waf1 has been shown to have pleiotropic functions that are cell-type specific [51, 52, 53]. Most recently, the p21/waf1 function was identified as a molecular barrier for HIV infection of stem cells . Zhang J et al have demonstrated a novel mechanism in which the anti-HIV effect of p21/waf1 was the result of its interaction with HIV-1 preintegration complex (PIC). Therefore, p21/waf1 was suggested to be a possible restriction factor, similar in function to the TRIM5 and APOBEC3G genes [22, 23, 24, 25, 26]. Consistent with this notion we have shown that the inactivated signaling pathways p53 and p21/waf1 by HIV-1 infection can be restored by a small molecule 9AA. Further, the 9AA-induced p21/waf1 was found to be recruited to HIV-1 PIC. Interestingly, we found the small molecule 9AA also inhibits the viral DNA integration step, which indicates that the drug 9AA is involved in the late stage of HIV-1 infection, rather than the early stages of infection.
In our current study, we have shown for the first time a functional restoration of the important signaling pathway(s) inactivated by HIV-1 infection using small chemical molecules. Further, our study also revealed a molecular mechanism by which the 9AA-induced inhibition of HIV-1 virus replication. It would be of great interest to carry out a future screening of a large number of chemically synthesized 9AA analogs, through which we may be able to identify more effective components in activating the p53 and p21/waf1 pathways, and in inhibiting virus replication at low concentrations. Therefore our results may provide a novel therapeutical arsenal for combating HIV-1 infection.
Materials and methods
Plasmids, drugs and antibodies
Flag-Tat plasmid was obtained from Dr. Hiscott J. (McGill, Montreal). Drug 9-aminoacridine (9-AA) and DMSO was purchased from Sigma (Cata.Nr. 06650). P44 peptide was synthesized from GenScript Corporation (Piscataway, USA). Anti-p53 and anti-p53ser15 were purchased from Cell Signaling; anti-FLAG was purchased from Sigma; anti-p21/waf1 and anti-Actin were purchased from Santa Cruz Inc.
Cell culture, transfections and drug treatments
ACH2, CEM and PBMC cells were maintained in RPMI-1640 medium supplemented with 10% FBS, L-glutamine (2 mM), and penicillin (100 U/ml)/streptomycin (100 μg/ml) (Quality Biological). For transfection of FLAG-Tat plasmids into ACH2 cells, five millions ACH2 cells were transfected with 5 μg of plasmid by nucleofection according to the manufacturer's protocol (Amaxa, Cologne, Germany). The cells were then incubated for 6 hrs before treatment with 9AA or DMSO as mock control. Twenty four hrs after drug or DMSO treatment, the cells were harvested for evaluation of the Tat expression and IP experiments with specific antibodies.
Cell viability assays
After the indicated time of drug treatment, the cells were harvested and stained by Trypan blue. The viable cell number was normalized with control group and the results were expressed as relative cell viability. To evaluate the effects of 9AA on long-term growth, we collected PHA+IL-2 activated PBMC cells at different time-points, 0, 6, 12 and 18 days and stained for viability.
Cells were harvested at 4°C and cell pellets were washed with Dulbecco's phosphate-buffered saline (PBS). Cell lysates were prepared as previously described . Five micrograms of Anti-MA (ABI Inc., Columbia, MD) were incubated with 2 mg of whole cell lysates overnight at 4°C with rotation. The overnight-incubated mixture was then cleared by centrifugation and Protein A/G beads (30% slurry) were added for 2 h at 4°C. The immunoprecipitated complex was washed with buffer K (150 mM KCl, 20 mM HEPES, pH 7.4, 5 mM MgCl2), then resuspended in SDS-PAGE loading laemmli buffer. Samples were separated on a 4–20% SDS/PAGE gel and subjected to western blot.
Western blotting analysis
For SDS-PAGE and western blotting of p53, p21/waf1 and Tat, total cellular proteins were prepared with ice-cold lysis buffer (50 mM Tris, 5 mM EDTA, 0.1% Triton X-100, 150 mM NaCl and mixed cocktail protease inhibitors). Cell debris was removed by centrifugation, the supernatants were collected and the protein concentrations were determined by protein quantification kit (Bio-Rad, CA). Protein samples were separated on 4–20% Tris-glycine gels (Invitrgen), and transferred on PVDF membranes. Anti-p53, anti-p53ser15 (Cell Signaling) and anti-p21/waf1 were used for immunodetection (Santa Cruz).
Reverse transcriptase assay was performed according to a standard procedure. In brief, 10 ul of cell free supernatant was incubated in RT buffer (0.2 M Tris-HCL, 0.2 M DTT, 0.2 M MgCl2, 0.2 M KCL) in the presence of 0.1% Triton X-100, PolyA template, PolyD(T) primer and 3HTTP overnight at 37°C. After incubation 5 ul of the reaction mix was spotted on a DEAE filter and allowed to dry. Excess 3HTTP was removed by four washes with 5% Na2HPO4 followed by rinsing with water. Incorporation of 3HTTP was measured using a scintillation counter. RT activity is measured as CPM according to the scintillation readout.
Quantitative real-time PCR
CEM (12D7) cells were infected with HIV-1 LAI and followed by 9AA or DMSO treatments. Cells were harvested at different time-points, 24, 48, 72 hrs. Total DNA was isolated by DNAzol® Genomic DNA Isolation Reagent according to their instruction, and analyzed by Real-Time PCR using the TaqMan method with primers and probes specific for late reverse transcripts. Products were amplified from 10 μl of DNA in 50 μl reactions containing 1 × TaqMan Universal PCR Master Mix, 300 nM primers and 100 nM probe with primers: FOR-LATE (5'-TGTGTGCCCGTCTGTTGTGT-3'), REV-LATE (5'-GAGTCCTGCGTCGAGAGAGC-3') and the probe (5'-/56-FAM/CAGTGGCGCCCGAACAGGGA/36-TAMTph/-3) (Integrated DNA Technologies, Inc).
ACH2 cells treated with 9AA or DMSO were harvested in vitro kinase assay. Kinase assay was performed after immunoprecipitatting with anti-cycle E ab from 2 mg of ACH2 or CEM cells treated with 9AA or DMSO as mock control. The kinase assay was performed according to method described previously . Phosphorylated substrate Histone H1 was resolved on 4–20% Tris-glycine gel, dried and then subjected to autoradiography (Packard Instruments, Wellesley, MA).
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