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1 Department of Research and Development, Nanchang Helioeast Science and Technology Co. Ltd, Nanchang, Jiangxi 330096, People's Republic of China
2 Department of Biomedical Sciences and the Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, People's Republic of China
Correspondence
Baoan Li
bali{at}xmu.edu.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: University of California, Irvine, CA 92697, USA. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Witvrouw et al., 2004). This therapy has changed the course of HIV infection dramatically. However, the rapid development of drug resistance has led to the emergence of HIV strains that are resistant to multiple anti-AIDS drugs (Biesert et al., 1991; St Clair et al., 1991; Najera et al., 1994; Menéndez-Arias, 2002). Furthermore, these drugs have limited or transient benefits due to their adverse side effects (Tözsér, 2001). Therefore, the discovery and characterization of new anti-HIV agents is required for alternative therapeutic approaches to overcome the shortcomings of the currently available drugs.
Rev is an essential regulatory HIV-1 protein that binds unspliced and incompletely spliced viral mRNAs and mediates the transport of these mRNAs from the nucleus into the cytoplasm for translation into viral proteins. Translocation of Rev from the nucleus to the cytoplasm is crucially important for its function and for virus replication (Kalland et al., 1994; Meyer & Malim, 1994). Blocking the Rev transport process would inhibit Rev function and this is a potential target for the development of antiviral therapies. In fact, several different strategies have been employed to block HIV-1 replication by inhibiting the Rev function (Matsukura et al., 1989; Bevec et al., 1992, 1996; Lee et al., 1992; Zapp et al., 1993; Duan et al., 1994). However, only a few small organic compounds have been reported to block Rev export from the nucleus (Wolff et al., 1997; Murakami et al., 2002). In this study, we screened 600 different extracts from medicinal plants by using a Rev transport assay and identified 1'S-1'-acetoxychavicol acetate (ACA) isolated from Alpinia galanga as a novel and effective Rev transport inhibitor. We tested this activity further and found that ACA could compete with leptomycin B (LMB) for chromosomal region maintenance 1 (CRM1) binding and blocked full-length HIV-1 RNA export from the nucleus to the cytoplasm. Furthermore, we demonstrated that ACA inhibited >80 % of HIV-1 replication in peripheral blood mononuclear cells (PBMCs) at a concentration of 4 µM and its antiviral activity was found to be synergistic when delivered with other antiretroviral agents.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Briefly, S. pombe was grown in thiamine-free minimal medium for 24 h and cells were seeded in 96-well plates. The screening samples were added to each well at a concentration of 100 µg ml–1 in 1 % DMSO and incubated at 37 °C for 3 h. The distribution of the GST–NLS–GFP–RevNES fusion protein was monitored by fluorescence microscopy.
Chemicals.
ACA was isolated from the rhizomes of greater galangal (A. galanga) that was collected in China. Greater galangal (100 g dry weight) was extracted with methanol. The extract was purified by silica-gel column chromatography and eluted with n-hexane/ethyl acetate (at ratios of 20 : 1, 10 : 1, 5 : 1 and 1 : 1) and chloroform/methanol (1 : 1). The n-hexane/ethyl acetate 5 : 1 fraction was purified further by reverse-phase HPLC [Cosmosil 5C18-AR, methanol/water (4 : 1)] to obtain a pure compound (1.5 g). The chemical structure of this pure compound (RF 0.4, silica gel, n-hexane/ethyl acetate 5 : 1) was identified as ACA by comparison of the spectral data of mass and nuclear magnetic resonance with that of reported compounds (De Pooter et al., 1985; Morita & Itokawa, 1988). Didanosine (ddI) was purchased from Sigma.
Cell culture and preparation of HIV-1 stock.
PBMCs were isolated from whole blood of healthy donors by density centrifugation with Ficoll-Hypaque (Sigma). PBMCs were cultured in RPMI 1640 containing 20 % fetal bovine serum (FBS) and 4 µg phytohaemagglutinin (PHA) ml–1 (Invitrogen). After 3 days, PHA-stimulated PBMCs were washed three times with PBS and cultured in RPMI 1640 containing 20 % FBS and 10 U interleukin 2 ml–1 (Boehringer Mannheim). Human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10 % FBS and 50 µg gentamicin ml–1. Fresh viral stocks were generated by transient transfection of 293T cells with plasmids pNL4-3 and 
SVJR-CSF bearing full-length infectious molecular clones of HIV-1 NL4-3 and JR-CSF, respectively, by calcium phosphate co-precipitation. Briefly, 100 µg plasmid DNA was mixed with 2 M CaCl2 and added to an equal volume of 2x HEPES-buffered saline. The DNA mixture was then incubated with 293T cells (seeded at 9x106 cells per T75 flask) in the presence of 25 µM chloroquine for 7 h and then washed away. Three days post-transfection, virus-containing supernatants were collected and frozen at –80 °C until needed.
HIV p24 ELISA.
An HIV-1 p24 ELISA was performed by using a commercially available kit (Perkin Elmer) according to the manufacturer's instructions. For measuring HIV-1 p24 antigen production in the supernatants, 100-fold dilutions of the supernatant were used. All ELISA measurements were performed in triplicate.
Antiviral assay.
Analysis of the antiviral activity of ACA on HIV-1 replication was based on inhibition of p24 viral antigen production. Briefly, PHA-activated PBMCs were grown in 24-well plates. After spinning (to further enhance attachment of the virus to the target cells; spinfection) with NL4-3 or JR-CSF virus, cells were washed twice in PBS and replaced by fresh culture medium. Different concentrations of ACA and/or the reverse transcriptase inhibitor ddI were added at various times post-infection. Five days later, supernatants were collected and HIV-1 p24 was detected by ELISA as described above.
Cell-viability assay.
Cell-viability assays were conducted with the CellTiter 96 Cell Proliferation assay system (Promega) according to the manufacturer's protocol. Briefly, PBMCs were plated into a flat-bottomed 96-well plate at a density of 105 cells per well in 100 µl medium and various concentrations of ACA (0.5, 1, 2, 3 and 4 µM) were added. After 48 h incubation, 2 ml methyltetrazolium salt (MTS) was mixed with phenazine methosulfate (100 µl) and 20 µl of the mixture was added to each well. The plate was incubated for 3 h at 37 °C and A490 was read on a spectrophotometer. The percentage of viable cells treated with ACA was normalized to untreated cells.
Competition assay.
293T cells (107) were cultured with 100 nM LMB (Sigma), ACA (0.5, 1, 2 or 4 µM) or 0.1 % ethanol for 2 h and then treated or not with 10 nM biotinylated LMB for 3 h. After the cells had been lysed with 0.1 % NP-40 in TBS [50 mM Tris/HCl (pH 7.4), 150 mM NaCl], the supernatant was prepared by centrifugation and treated with immobilized streptavidin-conjugated agarose beads (Sigma) in TBS under rotation for 24 h at 4 °C. Bound proteins were washed thoroughly and eluted by boiling in 30 µl SDS-PAGE sample buffer. Each eluted sample was separated by SDS-PAGE (10 % gel) and proteins were transferred to Immobilon PVDF membrane (Millipore). The membrane was probed with anti-CRM1 antibody (1 : 1000 dilution; Biocompare). Horseradish peroxidase-labelled secondary antibodies were detected by the Amersham Biosciences enhanced chemiluminescence system. As an internal control, the protein concentration of the supernatants was measured and the expression of 
-actin was also detected by Western blot analysis.
RNA extraction and quantitative analysis.
The cytoplasmic fraction was isolated by treatment with digitonin lysis buffer [50 mM HEPES/KOH (pH 7.5), 50 mM potassium acetate, 8 mM MgCl2, 2 mM EDTA, 50 µg digitonin ml–1] on ice for 10 min. The lysate was centrifuged for 5 min and the supernatant was collected as the cytoplasmic fraction. Pellets were resuspended in NP-40 lysis buffer [20 mM Tris/HCl (pH 7.5), 50 mM KCl, 10 mM NaCl, 1 mM EDTA, 0.5 % NP-40) and incubated on ice for 10 min. The resultant lysate was used as the nuclear fraction. Cytoplasmic RNA and nuclear RNA were extracted and purified from the cytoplasmic fraction and the nuclear fraction, respectively, by using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The amount of extracted RNA was quantified by measuring A260. RNA (1 µg) was treated with 1 U DNase I (Invitrogen) in a volume of 10 µl to remove contaminating DNA (room temperature for 10 min, 70 °C for 5 min). DNase I-treated RNA (300 ng) was reverse-transcribed by using a two-step reverse transcription kit (Applied Biosystems) in a final volume of 10 µl. Reverse transcription was performed for 60 min at 37 °C. The total cDNA volume of 10 µl was frozen until real-time quantitative PCR was performed. After thawing for PCR experiments, the cDNA was diluted in 90 µl distilled water and 5 µl diluted cDNA was used for each PCR. Real-time quantitative PCR was performed by using the ABI Prism 7700 Sequence Detection system (PE Applied Biosystems) for amplification and detection. PCR conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Each PCR was carried out in triplicate and contained 15 µl SYBR Green PCR master mix (Applied Biosystems) and 0.3 µM each gene-specific primer for human DBR1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a 30 µl reaction volume. The HIV-1-specific primers JRFL51 (5'-CTGCTAGAGATTTTCCACACTGAC-3'; nt 1204–1227) and JRFL31 (5'-GCTGCTTGATGTCCCCCCACTGTG-3'; nt 1356–1333) were used to detect full-length HIV-1 RNA. Copies of full-length HIV-1 RNA were normalized against copies of GAPDH (endogenous reference), amplified by using primers 5'-GGTGGTCTCCTCTGACTTCAA-3' (nt 840–860) and 5'-GTTGCTGTAGCCAAATTCGTTGT-3' (nt 966–944) specific for GAPDH.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Therefore, the n-hexane/ethyl acetate 5 : 1 fraction was purified further to obtain the bioactive compound ACA (Fig. 1b). This compound completely inhibited the export of the RevNES fusion protein from the nucleus at 5 µM in comparison with the DMSO control (Fig. 1c, d).
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). This result demonstrated that ACA blocked the replication of both X4 and R5 HIV-1 significantly. The inhibition activity of ACA was also observed in both NL4-3- and JR-CSF-infected MT-2 cells (data not shown).
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).
Effect of ACA in combination with ddI on HIV-1 replication
If ACA is able to target HIV-1 Rev export from the nucleus to the cytoplasm, it was expected that combination of ACA with another antiretroviral inhibitor, such as the reverse transcriptase inhibitor ddI or 3'-azido-3'-deoxythymidine (AZT), would be synergistic. To test this hypothesis, we evaluated the inhibition of NL4-3 (Fig. 3a) and JR-CSF (Fig. 3b) virus replication in PBMCs in the presence of various concentrations of ACA and ddI alone or in combination. The data showed that ACA plus ddI strengthened the inhibition of HIV-1 production significantly, compared with ACA or ddI alone. The same effect was also found by using AZT instead of ddI (data not shown). These results indicated that ACA acts synergistically with different target anti-HIV agents to inhibit HIV-1 replication.
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). Although previous studies have identified LMB as a strong inhibitor of Rev transport, it is not an acceptable candidate for anti-HIV drug development due to its severe cytotoxicity (Wolff et al., 1997). In contrast, our result suggests that ACA is a safer HIV-1 replication inhibitor.
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). Our studies determined that ACA inhibited the nuclear export of the HIV-1 RevNES protein and showed high-level inhibition of HIV-1 replication in PBMCs. We hypothesized that the interaction between ACA and CRM1 was probably the same as that with LMB.
To confirm this hypothesis, ACA was used in an in vitro competition assay with biotinylated LMB for CRM1 binding. 293T cells were cultured with 10 nM biotinylated LMB for 2 h, biotin-containing complexes were isolated by using streptavidin-conjugated agarose beads and bound proteins were analysed by SDS-PAGE followed by Western blotting. Western blot analysis using an anti-CRM1 antibody showed that CRM1 was detected when the cell culture contained biotinylated LMB or a lower concentration of ACA. However, when 4 µM ACA (as a competitor) or 0.1 µM LMB (as a control competitor) was added to the cell culture 2 h before the biotinylated LMB was added, CRM1 was not detected (Fig. 5). In the competition experiment, ACA was required at a higher concentration than LMB, but 0.1 µM LMB showed strong cytotoxicity. These results indicated that ACA competes with LMB for CRM1 binding in the cells.
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; Hadzopoulou-Cladaras et al., 1989; Malim et al., 1989). If ACA inhibits Rev function, the unspliced and partially spliced viral mRNAs would accumulate in the nucleus. Therefore, we determined the quantity of cytoplasmic and nuclear full-length viral RNA in host cells to confirm whether ACA contributed to Rev-specific inhibition.
In this experiment, PBMCs were infected with JR-CSF virus in the absence or presence of increasing concentrations of ACA. Five days after infection, HIV-1 RNA in the cytoplasm and nucleus was extracted and the amount of full-length RNA was determined by using the specific primer pair JRFL51 (nt 1204–1227) and JRFL31 (nt 1356–1333) by real-time quantitative RT-PCR. An internal control was performed in which an equal amount of RNA from each cytoplasmic and nuclear fraction was analysed by real-time quantitative RT-PCR using primer pairs specific for cellular GAPDH mRNA. We observed that unspliced HIV-1 RNA showed a dose-dependent accumulation in the nucleus as expected. When cells were treated with 0.5 µM ACA, the accumulation of viral nuclear unspliced RNA showed a slight increase, whilst 4 µM ACA led to a significant increase (1.9-fold) in comparison with the untreated control (Fig. 6a). On the other hand, ACA resulted in a dose-dependent reduction of full-length RNA in the cytoplasm (Fig. 6b). This result was consistent with the data obtained from the LMB competition assay and indicated that ACA inhibits HIV function by blocking Rev transport.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This protein is a potential target for the development of antiviral therapies. Currently reported Rev inhibitors target mainly the interaction between Rev and the Rev-responsive element (Ratmeyer et al., 1996; Zapp et al., 1997). In this paper, we reported that the low-molecular-mass compound ACA, isolated from the rhizomes of A. galanga, inhibited Rev transport completely in an in vitro yeast model and reduced HIV-1 replication by >80 % in PBMCs. To investigate the mechanism of inhibition, we performed a competition binding assay with biotinylated LMB to test whether ACA had the same target as LMB. Our results showed clearly that ACA competed with biotinylated LMB in binding with CRM1. This result suggested that, similar to LMB, ACA may inhibit HIV-1 replication by blocking Rev transport via the CRM1 pathway. It has been reported that the Rev transport inhibitor LMB was identified as blocking HIV-1 replication. The inhibitory mechanism suggested that LMB bound covalently and selectively to the thiol group of Cys-529 in CRM1 via its 
,-unsaturated 
-lactone (Kudo et al., 1999). Whether ACA binds to the same thiol group of Cys-529 or to another site on CRM1, and the type of functional group of ACA that is involved in its binding to CRM1, are currently under investigation.
ACA was first isolated from the rhizomes of A. galanga and found to prevent the growth of various fungi (Janssen & Scheffer, 1985). It was also reported that ACA showed anti-tuberculosis and anti-allergy activity (Palittapongarnpim et al., 2002; Matsuda et al., 2003; Yoshikawa et al., 2004). Furthermore, numerous studies have demonstrated that ACA suppresses the development of many tumours, such as skin cancer, oral cancer, colon cancer, liver cancer, bile-duct cancer and oesophageal cancer in vivo (Murakami et al., 1996; Ohnishi et al., 1996; Tanaka et al., 1997a, b; Kobayashi et al., 1998; Kawabata et al., 2000; Miyauchi et al., 2000), but the mechanism is less well understood. Recently, ACA was found to inhibit beta interferon mRNA expression and nuclear factor 
B activation in lipopolysaccharide-activated mouse peritoneal macrophages, resulting in inhibition of the production of nitric oxide (Ando et al., 2005). In this study, we have discovered a new function for ACA. ACA was able to compete with LMB, an inhibitor of Rev transport, in binding to CRM1, suggesting that ACA, similar to LMB, probably blocks Rev transport via CRM1-mediated export pathways. We examined and compared quantitatively the amount of unspliced HIV-1 RNA in the nucleus and cytoplasm in the presence and absence of ACA. The results showed that ACA was able to increase full-length RNA accumulation in the nucleus and reduce the amount of full-length RNA in the cytoplasm, which suggests that ACA is involved in the inhibition of Rev–RNA transport from the nucleus to the cytoplasm.
The currently available HAART uses two non-nucleoside agents and one nucleoside reverse transcriptase inhibitor or two non-nucleoside reverse transcriptase inhibitors and one protease inhibitor. The significant disadvantages associated with these therapies are severe drug side effects and viral escape mutants. As a Rev transport inhibitor that targets a different stage of the HIV-1 replication cycle, ACA may produce favourable interactions with other agents to overcome these issues. We observed the synergistic effect of ACA with the reverse transcriptase inhibitor ddI and with AZT. Despite the fact that the two drugs act at different stages of the HIV-1 life cycle, the reasons for the synergistic interaction are not clear. However, these results indicate that ACA may significantly enhance the antiviral activity with other anti-HIV-1 drugs and suggests that ACA possesses advantages when combined with some of the currently used nucleoside analogues.
In conclusion, the development of the low-cost, low-cytotoxicity Rev transport inhibitor ACA is a promising approach towards providing novel antiretroviral therapies. When combined with other anti-HIV agents, ACA is extremely effective in inhibiting HIV-1 replication.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 11 November 2005;
accepted 27 February 2006.
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