A synthetic HIV-1 Rev inhibitor interfering with the CRM1-mediated nuclear export - PubMed (original) (raw)
A synthetic HIV-1 Rev inhibitor interfering with the CRM1-mediated nuclear export
Dirk Daelemans et al. Proc Natl Acad Sci U S A. 2002.
Abstract
The HIV-1 Rev protein is an essential regulator of the HIV-1 mRNA expression that promotes the export of unspliced and partially spliced mRNA. The export receptor for the leucine-rich nuclear export signal (NES) of Rev has recently been recognized as CRM1. We identified a low molecular weight compound PKF050-638 as an inhibitor of HIV-1 Rev. This drug inhibits in a dose-dependent fashion Rev-dependent mRNA expression in a cellular assay for Rev function. We show that PKF050-638 is an inhibitor of the CRM1-mediated Rev nuclear export. By using a quantitative in vitro CRM1-NES cargo-binding assay, we could demonstrate that PKF050-638 disrupts CRM1-NES interaction. This mode of action is confirmed in cell culture because the drug reversibly interferes with the colocalization of CRM1 and Rev in the nucleolus of the cell. In addition, we prove that the inhibition is through direct interaction of the compound with Cys-539 of CRM1. These effects are similar to those of the known CRM1 inhibitor leptomycin B and suggest that the inhibitory effect of the compound is caused by binding to CRM1 at a similar site. The compound displayed strict structural requirements for its activity, as its enantiomer was inactive in all assays tested. These results show that we identified a drug that interferes with the CRM1-mediated nuclear export of Rev through inhibition of the CRM1-NES complex formation. The reversibility of its binding to CRM1 and its availability through chemical synthesis could make it useful for studying CRM1-mediated export pathways.
Figures
Fig 1.
Chemical formulae of PKF050-638 and its inactive enantiomer PKF050-637. *, Asymmetric carbon.
Fig 2.
Effect of PKF050-638 on the localization of Rev14-GFP in living cells. HLtat cells were transfected with a plasmid expressing Rev14-GFP, and 24 h later, they were treated with PKF050-638 or LMB. Two hours after the addition of drugs, the cells were analyzed by confocal laser scan microscopy. Rev14-GFP localized in the cytoplasm but is able to shuttle between cytoplasm and nucleus (A). Addition of 5 μM PKF050-638 (B) or 50 nM LMB (C) resulted in predominantly nuclear/nucleolar localization of the mutant.
Fig 3.
Effect of PKF050-638 on the Rev NES in living cells. (A) Tat-GFP-NES accumulates in the cytoplasm of cells. (B) Treatment of the cells with 5 μM PKF050-638 induces nuclear retention of Tat-GFP-NES within 2 h.
Fig 4.
Reversible effect of PKF050-638 on the localization of Rev14-GFP in living cells. Four hours after drug treatment, the drugs were washed out, and cells were analyzed by confocal scan microscopy after overnight incubation. Rev14-GFP in untreated localizes in the cytoplasm as shown in Fig. 2_A_. (A) Cells incubated with 7.5 μM PKF050-638 and washed resulted in predominant relocalization of the Rev14-GFP in the cytoplasm, whereas (B) in most cells treated with 100 nM LMB and washed, the Rev14-GFP stayed in the nucleoli. Before addition of compound (0 h), the Rev14-GFP in all cells was cytoplasmatic (0% of cells showed GFP in nucleoli). After the addition of either compound, all of the cells had Rev14-GFP in the nucleoli. The cells then were washed, and in 50% of cells incubated with LMB, Rev14-GFP still localized in the nucleoli, whereas Rev14-GFP could be detected in the nucleolus in only 14% of cells incubated with PKF050-638.
Fig 5.
Colocalization of hCRM1-GFP with Rev-BFP in HeLa cells. HeLa cells were cotransfected with phCRM1-GFP and pRev-BFP and analyzed by fluorescence microscopy. For GFP, emitted fluorescence was detected with a 510- to 540-nm band-pass filter (Upper), and BFP expression was analyzed with a 400–440 nm band-pan filter (Lower). Rev-BFP is localized into nucleoli (Nu in D), whereas hCRM1-GFP is found at the nuclear rim of the cells not expressing Rev-BFP (NE in A). In cells coexpressing Rev-BFP and hCRM1-GFP, the latter colocalizes with Rev-BFP in the nucleoli (Nu in A). Thirty and 60 minutes after the addition of 7.5 μM PKF050-638, the colocalization of hCRM1-GFP with Rev-BFP disappeared (B and C), whereas Rev-BFP stays in the nucleoli (E and F).
Fig 6.
Quantitative analysis of NES-CRM1 affinity in vitro using the RanGAP hydrolysis assay. Inhibitory effect of LMB (1 μM; ▵), PKF050-638 (330 μM; ▴), PKF050-637 (330 μM; ×), and no drug (♦) on CRM1-NS2 (A) or CRM1-PKI (B) binding as measured from CRM1-dependent protection of Rna1p-stimulated GTP hydrolysis on Ran, as a function of increasing concentrations of CRM1. In all series, NS2 or PKI peptides are present at 40 and 200 μM, respectively.
Fig 7.
Analysis of the effect of PKF050-638 on WT S. cerevisiae (A) and a Crm1pT539C-expressing strain (B). Overnight cultures of the two strains were diluted until OD600 ≈ 0.1 and were grown at 30°C in the presence of 80 nM LMB (*), different concentrations PKF050-638 (▵, 240 μM; +, 48 μM) and PKF050-637 (□, 240 μM; −, 48 μM), or with no drug (×). The evolution of the optical density at 600 nm is plotted.
References
- Meyer B. E. & Malim, M. H. (1994) Genes Dev. 8, 1538-1547. - PubMed
- Stauber R. H., Afonina, E., Gulnik, S., Erickson, J. & Pavlakis, G. N. (1998) Virology 251, 38-48. - PubMed
- Szilvay A. M., Brokstad, K. A., Boe, S. O., Haukenes, G. & Kalland, K. H. (1997) Virology 235, 73-81. - PubMed
- Fornerod M., Ohno, M., Yoshida, M. & Mattaj, I. W. (1997) Cell 90, 1051-1060. - PubMed
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