Inhibition of human immunodeficiency virus Rev and human T-cell leukemia virus Rex function, but not Mason-Pfizer monkey virus constitutive transport element activity, by a mutant human nucleoporin targeted to Crm1 - PubMed (original) (raw)

Inhibition of human immunodeficiency virus Rev and human T-cell leukemia virus Rex function, but not Mason-Pfizer monkey virus constitutive transport element activity, by a mutant human nucleoporin targeted to Crm1

H P Bogerd et al. J Virol. 1998 Nov.

Abstract

The hypothesis that the cellular protein Crm1 mediates human immunodeficiency virus type 1 (HIV-1) Rev-dependent nuclear export posits that Crm1 can directly interact both with the Rev nuclear export signal (NES) and with cellular nucleoporins. Here, we demonstrate that Crm1 is indeed able to interact with active but not defective forms of the HIV-1 Rev NES and of NESs found in other retroviral nuclear export factors. In addition, we demonstrate that Crm1 can bind the Rev NES when Rev is assembled onto the Rev response element RNA target and that Crm1, like Rev, is a nucleocytoplasmic shuttle protein. Crm1 also specifically binds the Rev NES in vitro, although this latter interaction is detectable only in the presence of added Ran . GTP. Overexpression of a truncated, defective form of the nucleoporin Nup214/CAN, termed DeltaCAN, that retains Crm1 binding ability resulted in the effective inhibition of HIV-1 Rev or human T-cell leukemia virus Rex-dependent gene expression. In contrast, DeltaCAN had no significant affect on Mason-Pfizer monkey virus constitutive transport element (MPMV CTE)-dependent nuclear RNA export or on the expression of RNAs dependent on the cellular mRNA export pathway. As a result, DeltaCAN specifically blocked late, but not early, HIV-1 gene expression in HIV-1-infected cells. These data strongly validate Crm1 as a cellular cofactor for HIV-1 Rev and demonstrate that the MPMV CTE nuclear RNA export pathway uses a distinct, Crm1-independent mechanism. In addition, these data identify a novel and highly potent inhibitor of leucine-rich NES-dependent nuclear export.

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Figures

FIG. 1

FIG. 1

Specific interaction between hCrm1 and functional leucine-rich NESs detected in the mammalian two-hybrid assay. (A) The hCrm1 protein was expressed fused to the GAL4 DNA binding domain, while the indicated retroviral proteins were expressed as VP16 transcription activation domain (AD) fusions. COS cell cultures (in 100-mm-diameter dishes) were transfected by the DEAE-dextran procedure, using 1 μg of the pG6(−31)HIVLTRΔTAR reporter plasmid, 0.5 μg of the pGAL4-Crm1 expression plasmid, 1 μg of the relevant VP16 fusion protein expression plasmid, and 1 μg of the pBC12/CMV/βgal internal control plasmid. Transfected cells were harvested at ∼48 h posttransfection and analyzed for CAT and β-Gal expression levels as previously described (4, 18). (B) As for panel A except that COS cell cultures (on 35-mm-diameter dishes) were transfected by the calcium phosphate procedure, using 2 μg of GAL4-Crm1, 2 μg of each VP16 fusion expression plasmid, 0.5 μg of the pG6(−31)HIVLTRΔTAR indicator, and 0.1 μg of pBC12/CMV/βgal. The indicated mutants of the Rev NES (75-LPPLERLTLD-84) have been described elsewhere (31) and are as follows: M10, Leu 78 and Glu 79 to Asp and Leu, respectively; M19, Pro 77 to Asp; M21, Leu 81 and Thr 82 to Asp and Leu; M22, Leu 83 and Asp 84 to Asp and Leu; M27, Leu 78 to Ala; M29, Leu 83 to Ala; M32, Leu 78, Leu 81, and Leu 83 all to Ala. The only active Rev mutant is M19. These data are representative of three separate transfection experiments.

FIG. 2

FIG. 2

hCrm1 binds to Rev when Rev is bound to the RRE. The pSLIIB/CAT indicator construct contains an HIV-1 LTR in which the TAR RNA binding site for Tat has been substituted by the RRE SLIIB RNA binding site for Rev. When a Crm1-Tat fusion protein is coexpressed with Rev, the pSLIIB/CAT indicator is activated by recruitment of Tat to the SLIIB RNA target by the Crm1-Rev protein-protein interaction. This effect is not observed when either protein is expressed alone or if the Rev NES (M10) or the Rev RNA binding site (M5) is mutated. 293T cells (in 35-mm-diameter dishes) were transfected by the calcium phosphate procedure with 500 ng of pSLIIB/CAT, 1,000 ng of pcRev, pM10, or pM5, 500 ng of pBC12/Crm1/Tat, and 100 ng of pBC12/CMV/βgal. The parental pBC12/CMV plasmid was substituted where necessary as a negative control. The data are representative of four separate transfection experiments.

FIG. 3

FIG. 3

The Rev NES forms a ternary complex with hCrm1 and Ran · GTP in vitro. The hCrm1 protein was synthesized in vitro, in the presence of [35S]methionine, using a rabbit reticulocyte coupled transcription-translation system (Promega). The labeled proteins were then divided into two equal aliquots, and one was supplemented with recombinant Ran · GTP. The reaction mixtures were then again divided into two equal parts, and each fraction was loaded onto affinity columns bearing recombinant GST-Rev or the GST-M10 NES mutant. Bound proteins were eluted and resolved by SDS-PAGE. The data are representative of two separate protein interaction experiments.

FIG. 4

FIG. 4

hCrm1 is a nucleocytoplasmic shuttle protein. Recombinant purified GST-Crm1 was mixed with a rabbit IgG tracer and then injected into one nucleus of a binuclear HeLa cell. While the IgG tracer remains behind (A), the Crm1 fusion protein relocalizes to the other nucleus, and in particular to the nuclear membrane, as well as to the cytoplasm (B).

FIG. 5

FIG. 5

The ΔCAN protein is a specific inhibitor of leucine-rich NES-dependent mRNA expression. (A) The pDM128/CMV Rev indicator construct contains the cat gene and the HIV-1 RRE located in an intron defined by HIV-1 splice sites (26, 31). Efficient CAT expression is therefore dependent on the Rev-induced nuclear export of an unspliced form of this RNA. Similar indicator plasmids containing the VMV RRE, the HTLV-1 RXRE, or the MPMV CTE were also tested. A pDM128/CMV derivative lacking any inserted RNA target site (pDM128/CMV/PL) served as a negative control for pDM128/CTE. 293T cells (in 35-mm-diameter dishes) were transfected with 50 ng of the relevant HIV-1 Rev, VMV Rev, or HTLV-1 Rex reporter plasmid, 50 ng of the relevant effector plasmid, 100 ng of the pBC12/CMV/βgal internal control plasmid, and 500 ng of pBC12/CMV/ΔCAN. The pBC12/CMV negative control plasmid was added to maintain a level of 1,050 ng of DNA per transfection. Plasmid pDM128/CTE or pDM128/CMV/PL was transfected at 500 ng per culture, together with 100 ng of pBC12/CMV/βgal and 500 ng of either pBC12/ΔCAN or pBC12/CMV. The data are representative of three separate transfection experiments. (B) As for panel A except that the pBC12/ΔCAN expression plasmid was introduced at increasing levels, up to 500 ng per transfection. Total transfected DNA was held constant by substitution of pBC12/CMV. ⧫, CAT activity induced by the pDM128/CMV/PL negative control for pDM128/CTE; ▵, CAT activity induced by pDM128/CMV in the absence of HIV-1 Rev. (C) Northern analysis of cytoplasmic unspliced (Unspl.) and spliced (Spl.) RNA expression in 293T cells transfected with the pDM128/CMV indicator construct and plasmids expressing HIV-1 Rev and ΔCAN, as described for panel A. The parental pBC12/CMV plasmid served as a negative control.

FIG. 5

FIG. 5

The ΔCAN protein is a specific inhibitor of leucine-rich NES-dependent mRNA expression. (A) The pDM128/CMV Rev indicator construct contains the cat gene and the HIV-1 RRE located in an intron defined by HIV-1 splice sites (26, 31). Efficient CAT expression is therefore dependent on the Rev-induced nuclear export of an unspliced form of this RNA. Similar indicator plasmids containing the VMV RRE, the HTLV-1 RXRE, or the MPMV CTE were also tested. A pDM128/CMV derivative lacking any inserted RNA target site (pDM128/CMV/PL) served as a negative control for pDM128/CTE. 293T cells (in 35-mm-diameter dishes) were transfected with 50 ng of the relevant HIV-1 Rev, VMV Rev, or HTLV-1 Rex reporter plasmid, 50 ng of the relevant effector plasmid, 100 ng of the pBC12/CMV/βgal internal control plasmid, and 500 ng of pBC12/CMV/ΔCAN. The pBC12/CMV negative control plasmid was added to maintain a level of 1,050 ng of DNA per transfection. Plasmid pDM128/CTE or pDM128/CMV/PL was transfected at 500 ng per culture, together with 100 ng of pBC12/CMV/βgal and 500 ng of either pBC12/ΔCAN or pBC12/CMV. The data are representative of three separate transfection experiments. (B) As for panel A except that the pBC12/ΔCAN expression plasmid was introduced at increasing levels, up to 500 ng per transfection. Total transfected DNA was held constant by substitution of pBC12/CMV. ⧫, CAT activity induced by the pDM128/CMV/PL negative control for pDM128/CTE; ▵, CAT activity induced by pDM128/CMV in the absence of HIV-1 Rev. (C) Northern analysis of cytoplasmic unspliced (Unspl.) and spliced (Spl.) RNA expression in 293T cells transfected with the pDM128/CMV indicator construct and plasmids expressing HIV-1 Rev and ΔCAN, as described for panel A. The parental pBC12/CMV plasmid served as a negative control.

FIG. 6

FIG. 6

The ΔCAN protein inhibits Rev NES function. COS cells were transfected (9) with the wild-type HIV-1 Rev expression plasmid pcRev together with a second plasmid expressing ΔCAN or with plasmid pBC12/CMV as a negative control. At ∼48 h after transfection, some cultures were treated with actinomycin D (Act D; 5 μg/ml) for 4 h, and all cultures were then fixed by using 3% paraformaldehyde in PBS. The subcellular localization of the Rev protein was determined by immunofluorescence, as previously described (28), using a polyclonal rabbit anti-Rev antiserum and FITC-conjugated goat anti-rabbit IgG.

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