A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1 - PubMed (original) (raw)

A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1

Min-Hsuan Lin et al. Retrovirology. 2014.

Abstract

Background: Previously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex.

Results: To seek a possible mechanism that could explain how Nullbasic inhibits Rev activity, we used a proteomics approach to identify host cellular proteins that interact with Nullbasic. Forty-six Nullbasic-binding proteins were identified by mass spectrometry including the DEAD-box RNA helicase, DDX1. To determine the effect of DDX1 on Nullbasic-mediated Rev activity, we performed cell-based immunoprecipitation assays, Rev reporter assays and bio-layer interferometry (BLI) assays. Interaction between DDX1 and Nullbasic was observed by co-immunoprecipitation of Nullbasic with endogenous DDX1 from cell lysates. BLI assays showed a direct interaction between Nullbasic and DDX1. Nullbasic affected DDX1 subcellular distribution in a Rev-independent manner. Interestingly overexpression of DDX1 in cells not only restored Rev-dependent mRNA export and gene expression in a Rev reporter assay but also partly reversed Nullbasic-induced Rev subcellular mislocalization. Moreover, HIV-1 wild type Tat co-immunoprecipitated with DDX1 and overexpression of Tat could rescue the unspliced viral mRNA levels inhibited by Nullbasic in HIV-1 expressing cells.

Conclusions: Nullbasic was used to further define the complex mechanisms involved in the Rev-dependent nuclear export of the 9 kb and 4 kb viral RNAs. All together, these data indicate that DDX1 can be sequestered by Nullbasic leading to destabilization of the Rev nucleocytoplasmic transport complex and decreased levels of Rev-dependent viral transcripts. The outcomes support a role for DDX1 in maintenance of a Rev nuclear complex that transports viral RRE-containing mRNA to the cytoplasm. To our knowledge Nullbasic is the first anti-HIV protein that specifically targets the cellular protein DDX1 to block Rev's activity. Furthermore, our research raises the possibility that wild type Tat may play a previously unrecognized but very important role in Rev function.

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Figures

Figure 1

Figure 1

Proteomic strategy for identifying Nullbasic-interacting proteins from HeLa-Nullbasic-mCherry cells. A. Overview of the proteomic approach for isolating Nullbasic (NB) binding proteins from cell nuclear extracts. B. Schematic diagrams of the VLP expression plasmids and an overview of the methods used to generate lentiviral VLPs. pCMV∆R8.91, pCMV-VSV-G and pLOX/CW-Nullbasic-FLAG-mCherry or pLOX/CW-FLAG-mCherry were co-transfected into HEK293T cells to produce VLPs containing either Nullbasic-FLAG-mCherry lentivector genome (VLP-NB-mCherry) or the control FLAG-mCherry genome (VLP-FLAG-mCherry). VLP-NB-mCherry and VLP-FLAG-mCherry were then transduced into HeLa cells in order to create cell lines stably expressing either the NB-mCherry proteins or FLAG-mCherry proteins. C. Nuclear protein lysates expressing NB-mCherry or FLAG-mCherry were separated by SDS-PAGE and protein bands were stained with Coomassie dye.

Figure 2

Figure 2

Interactions of DDX1, DDX3 and DDX17 with Nullbasic in vivo . HEK293T cells were transfected with either empty vector (pcDNA 3.1) or plasmid expressing NB-FLAG. The FLAG-tagged proteins and their binding partners were immunoprecipated with anti-FLAG beads. Total cell lystes and immunoprecipated proteins were separated by SDS-PAGE and target proteins were detected using anit-FLAG, anti-DDX1, anti-DDX3 and anti-DDX17 antibodies. The anti-CDK9 antibody was used to detect endogenous CDK9 as a positive control for NB interaction. The figure is representative of 3 independent experiments.

Figure 3

Figure 3

Overexpression of DDX1 in HeLa cells rescues the Rev/RRE-dependent reporter gene expression suppressed by Nullbasic. A. Schematic maps of pGag-RRE and pGag-CTE plasmids. B. Left: HeLa cells were transfected with appropriate expression plasmids, including pGag-RRE (500 ng), pRSV-Rev (10 ng), Nullbasic (NB, 500 ng), HA-DDX1(1000 ng), HA-DDX3 (1000 ng), DDX17 (1000 ng) and empty plasmid (pcDNA3-HA, 1000 g). Right: HeLa cells were transfected pGag-CTE (500 ng) with pRSV-Rev (10 ng), Nullbasic (NB) (500 ng), HA-DDX1(1000 ng) or empty vector (pcDNA3-HA, 1000 ng). A pcDNA3 plasmid was used to normalize the total amount of transfected plasmids and a luciferase expression plasmid was included to monitor transfection efficiency. After 24 h transfection, the cellular lysates were collected for assay of HIV-1 capsid level and luciferase activity. The p24 production was normalized to the luciferase reporter activity and expressed as a percentage relative to cells transfected with pGag-RRE with pRSV-Rev (left) or pGag-CTE with pRSV-Rev (right). Columns represent the means and standard deviations of transfections performed in triplicate. The experiment was performed 3 times with similar results.

Figure 4

Figure 4

Nullbasic alters the subcellular localization of DDX1. A. HeLa cells were transfected with empty vector alone (pcDNA3.1, row 1), MYC-Rev alone (row 2), Nullbasic (NB)-mCherry alone (row 3 a) or MYC-Rev with NB-mCherry (row 4). Fixed cells were immunostained with anti-MYC (green) and anti-DDX1 (magenta, upper panels) and were visualized alone with NB-mCherry (red) by fluorescence microscopy. Nuclei were stained with DAPI. The overlay panels show the merge of the Rev panel with DDX1 (row 1, 2, and 4) and the NB-mCherry panel with DDX1 panel (row 3). Images are representative of at least five fields selected randomly from three independent experiments. B. Quantification of nuclear/cytoplasmic fluorescence ratios(Fn/c) for DDX1as described in Methods. Mean of Fn/c ± SD were calculated from 60 HeLa cells from 3 independent experiments.

Figure 5

Figure 5

Overexpression of DDX1 in HeLa cells restores Rev nucleolar localization disrupted by Nullbasic. HeLa cells were transfected with HA-DDX1 alone (row 1), MYC-Rev with HA-DDX1 (row 2). Nullbasic (NB)-mCherry with HA-DDX1 (row 3) or MYC-Rev with HA-DDX1 and NB-mCherry (row 4). Fixed cells were immunostained with anti-MYC (green) and anti-HA (magenta) antibodies and were visualized alone with NB-mCherry (red) by fluorescence microscopy. Nuclei were stained with DAPI. The overlay panels show the merge of the MYC-Rev panel with the HA-DDX1 panels (row 1, 2, and 4) and the NB-mCherry panel with HA-DDX1 panel (row 3). Images are representative of at least five fields selected randomly from three independent experiments.

Figure 6

Figure 6

DDX1 directly interacts with Nullbasic in vitro . The Octet Red system was used to measure binding events between Nullbasic and DDX1. A. BLI sensograms. Biotinylated recombinant Nullbasic-FLAG-V5-6 × His was bound to a streptavidin biosensor and applied to solutions containing 3.3 nM (green), 10 nM (light blue), 30 nM (red) or 90 nM (dark blue) of human recombinant Myc-DDK-tagged DDX1. B. The probe was also exposed to DDX5 solutions of the same concentration or with BSA at 90 nM as negative controls. The BLI experiment was repeated 3 times with similar results and a representative sensogram is shown.

Figure 7

Figure 7

DDX1 forms different protein complexes with Nullbasic and Rev. A. HEK293T cells were transfected with either empty vector (pcDNA 3.1) alone, Nullbasic (NB)-FLAG alone, Rev alone or co-transfect NB-FLAG with Rev as indicated. Endogenous DDX1, NB-FLAG and their binding partners were immunoprecipated with anti-DDX1 and anti-FLAG beads. Cell lystes and immunoprecipated proteins were separated by SDS-PAGE and target proteins were detected using anti-FLAG, anti-DDX1 and anti-Rev antibodies. B. HEK293T cells were transfected with empty vector or Tat-FLAG as indicated. Target proteins were detected using anti-FLAG, anti-DDX1 after immunoprecipitation using anti-DDX1, anti-FLAG beads or with beads alone. The figures are representative of 3 independent experiments.

Figure 8

Figure 8

Overexpression of wild type Tat rescues the unspliced viral mRNA levels suppressed by Nullbasic in HIV-1 expressing cells. Total RNA was extracted from HEK293T cells transfected to express the HIV-1 infectious provirus, pGCH, and co-transfected at a 1:2 molar ratio with either empty vector (“No Tat”) or plasmids expressing Nullbasic-FLAG (“NB”), Tat-FLAG (“Tat”), or Nullbasic-FLAG with Tat-FLAG (“NB + Tat”). The levels of total (black columns) and unspliced (gray columns) HIV-1 mRNAs were assayed by quantitative RT-PCR using primers specific for total and unspliced viral mRNA. Results represent the means and standard deviations of triplicate assays from 3 independent experiments, with unspliced viral mRNA levels expressed relative to total viral mRNA levels. Significant differences between data were determined using Welch’s _t_-test against a two-tailed distribution, the p values from which are indicated.

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References

    1. Pollard VW, Malim MH. The HIV-1 Rev protein. Annu Rev Microbiol. 1998;52:491–532. doi: 10.1146/annurev.micro.52.1.491. - DOI - PubMed
    1. Venkatesh LK, Mohammed S, Chinnadurai G. Functional domains of the HIV-1 rev gene required for trans-regulation and subcellular localization. Virology. 1990;176(1):39–47. doi: 10.1016/0042-6822(90)90228-J. - DOI - PubMed
    1. Henderson BR, Percipalle P. Interactions between HIV Rev and nuclear import and export factors: the Rev nuclear localisation signal mediates specific binding to human importin-beta. J Mol Biol. 1997;274(5):693–707. doi: 10.1006/jmbi.1997.1420. - DOI - PubMed
    1. Szebeni A, Mehrotra B, Baumann A, Adam SA, Wingfield PT, Olson MO. Nucleolar protein B23 stimulates nuclear import of the HIV-1 Rev protein and NLS-conjugated albumin. Biochemistry (Mosc) 1997;36(13):3941–3949. doi: 10.1021/bi9627931. - DOI - PubMed
    1. Truant R, Cullen BR. The arginine-rich domains present in human immunodeficiency virus type 1 Tat and Rev function as direct importin beta-dependent nuclear localization signals. Mol Cell Biol. 1999;19(2):1210–1217. - PMC - PubMed

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