HEXIM1-Tat chimera inhibits HIV-1 replication - PubMed (original) (raw)

HEXIM1-Tat chimera inhibits HIV-1 replication

Marie Leoz et al. PLoS Pathog. 2018.

Abstract

Transcription of HIV provirus is a key step of the viral cycle, and depends on the recruitment of the cellular positive transcription elongation factor b (P-TEFb) to the HIV promoter. The viral transactivator Tat can displace P-TEFb from the 7SK small nuclear ribonucleoprotein, where it is bound and inactivated by HEXIM1, and bring it to TAR, which allows the stalled RNA polymerase II to transition to successful transcription elongation. In this study, we designed a chimeric inhibitor of HIV transcription by combining functional domains from HEXIM1 and Tat. The chimera (HT1) potently inhibited gene expression from the HIV promoter, by competing with Tat for TAR and P-TEFb binding, while keeping the latter inactive. HT1 inhibited spreading infection as well as viral reactivation in lymphocyte T cell line models of HIV latency, with little effect on cellular transcription and metabolism. This proof-of-concept study validates an innovative approach to interfering with HIV transcription via peptide mimicry and competition for RNA-protein interactions. HT1 represents a new candidate for HIV therapy, or HIV cure via the proposed block and lock strategy.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. A HEXIM1-Tat fusion peptide inhibits gene expression from the HIV promoter.

A. Structure of the HEXIM1-Tat fusion peptides used in the study. The functional domains used from HEXIM1 and Tat include a HEXIM1 Arginine Rich Motif (ARM, black box, residues 150–162) that binds RNA, a HEXIM1 inhibitory domain (ID, light grey box, residues 200–211) that inhibits CDK9 through a PYNT motif, and Tat transactivation domain (AD, dark grey box, residues 1–48) that binds to P-TEFb. The conceptual schemes are not drawn to scale. B. Transient expression of HT1 inhibits Tat-induced LTR-driven Luc expression. Upper panel: a schema depicts the reporter assay. A luciferase reporter gene (Luc) is under the control of the HIV promoter (P-HIV) and can be activated by ectopically expressed Tat. We use a Luc assay to titrate the inhibitory effect of HT1-3 on P-HIV transactivation by Tat. Middle panel: increasing amounts of m:HT1, m:HT2 or m:HT3 expressing plasmid (pHT) were co-transfected in 293T cells with a plasmid (pTat) expressing f:Tat and another (pLTR-Luc) expressing Luc under the control of the HIV promoter. Luc activity was plotted as % activity relative to control (empty vector used instead of pHT), depending on the transfected pHT : pTat ratio. Error bars in the graph represent standard deviation from triplicate experiments. Lower panel: expression levels of m:HT1 and f:Tat were confirmed by WB using anti-Myc and anti-Flag Abs. Housekeeping protein GAPDH was used as loading control. C. Transient expression of HT1 inhibits Luc expression from HIV-1 NL43ΔenvLuc. Left panel: a schema depicts the reporter assay. Luc is inserted in a replication-defective HIV-1 molecular clone (pNL43ΔenvLuc) in which Luc and Tat are under the control of the HIV promoter (P-HIV). We use a Luc assay to titrate the inhibitory effect of HT1 on P-HIV transactivation by Tat. Right panel: increasing amounts of pHT1 were co-transfected in 293T cells with pNL43ΔenvLuc. Luc activity was plotted as % activity relative to control (empty vector used instead of pHT), depending on the transfected amounts of pHT1. Error bars in the graph represent standard deviation from triplicate experiments.

Fig 2. HT1 prevents Tat from bringing active P-TEFb to TAR.

A. HT1 binds to P-TEFb and competes with Tat for P-TEFb binding. m:HT1 and/or f:Tat were transiently expressed in 293T cells. Cell lysates were used for immunoprecipitation (IP) using anti-Myc Ab (upper panel, lanes 2–7), anti-Flag Ab (middle panel, lanes 2–7), or control IgG (upper and middle panels, lane 1). Input lysates (lower panel) and immunoprecipitates were submitted to SDS-PAGE and WB using anti-CycT1, anti-Myc, anti-Flag and anti-actin Abs. B. HT1 inhibits the kinase activity of P-TEFb subunit CDK9. m:HT1 and/or f:Tat were transiently expressed in 293T cells. Cell lysates were used for IP using anti-Myc Ab (lanes 2–4), or control IgG (lane 1). Immunoprecipitates were incubated with ATP and recombinant GST-CTD proteins for in vitro kinase assay. Total GST-CTD was detected using anti-GST Ab and phosphorylated GST-CTD (CTD-P) was detected using anti-Ser2P Ab. m:HT1 and m:Tat were detected using anti-Myc Ab. Six replicate experiments were performed, and mean relative kinase activities are shown in the bar graph to the right. C. HT1 binds to TAR. m:HT1 or m:Tat (or empty vector, EV, as a control) was transiently co-expressed with TAR RNA-expressing pU16TAR in 293T cells. Cell lysates were used for IP using anti-Myc Ab or control IgG. RNA was purified from the immunoprecipitates and submitted to RT-qPCR using TAR-specific primers (upper left panel). Relative TAR enrichment was calculated as qPCR count using anti-Myc Ab minus using IgG, and normalized to EV. Error bars represent standard deviation from triplicate qPCR assays. Input lysates were submitted to SDS-PAGE and WB using anti-Myc and anti-Actin Abs (lower left panel). The amounts and standard deviations of immunoprecipitated TAR RNA (from the upper left panel) were normalized to the respective amount of m:HT1 or m:Tat detected in the input lysate (right panel). D. HT1 competes with Tat for TAR binding. m:HT1 and/or f:Tat were transiently co-expressed with TAR RNA-expressing pU16TAR in 293T cells. Cell lysates were used for IP using anti-Myc Ab or control IgG. RNA was purified from the immunoprecipitates and submitted to RT-qPCR using TAR-specific primers (upper panel). Relative TAR enrichment was calculated as qPCR count using anti-Myc Ab minus using IgG, and normalized to EV. Error bars represent standard deviation from triplicate qPCR assays. Input lysates were submitted to SDS-PAGE and WB using anti-Myc, anti-Flag and anti-Actin Abs (lower panel).

Fig 3. Expression of HT1 doesn’t impair cell gene expression and growth.

A. Transient expression of HT1 doesn’t impair P-TEFb dependent expression of endogenous HEXIM1 protein in 293T cells. Increasing amounts of m:HT1 were transiently expressed in 293T cells. Cell lysates were submitted to SDS-PAGE and WB using anti-Myc and anti-HEXIM1 Abs. B. Transient expression of HT1 doesn’t impair P-TEFb dependent transcription of endogenous hexim1 gene in 293T cells. Increasing amounts of m:HT1 were transiently expressed in 293T cells. Cellular RNA was purified and submitted to RT-qPCR using HEXIM1 and GAPDH specific primer pairs. The amount of HEXIM1 mRNA was normalized to that of GAPDH. Error bars represent standard deviation from triplicate experiments. C. Transient expression of HT1 doesn’t impair global gene expression in 293T cells. HT1 was transiently expressed in 293T cells. Cellular RNA was purified from these cells as well as from control 293T cells and CycT1 KO 293T cells, and submitted to mRNAseq. The upper panel shows Log2 fold change difference in gene expression between HT1-expressing cells and control cells, ranked by mean of normalized cell counts. Lower panel shows the same analysis for CycT1 KO cells relative to control. Red dots correspond to genes that are differentially expressed in HT1-expressing cells or in CycT1 KO cells compared to control (padj<0,1). Each condition was tested in triplicate experiments. D. Stable expression of HT1 in CEM, MOLT4 and MT4 cells 3f:HT1 was stably expressed in CEM, MOLT4, or MT4 cells (C-HT, MO-HT, MT-HT, respectively). Cell lysates were submitted to SDS-PAGE and WB using anti-Flag and anti-GAPDH Abs. E. Stable expression of HT1 doesn’t impair cell growth of C-HT, MO-HT and MT-HT cells. 100,000 cells were seeded in 2 mL RPMI with 10% FBS, and with 1 μg/mL puromycin for HT-expressing cells. Total viable cell count was performed at days 0–4 to assess cell growth. Error bars represent standard deviation from triplicate experiments.

Fig 4. Stable expression of HT1 inhibits HIV reactivation.

A. Stable expression of HT1 inhibits HIV reactivation from 2D10 cells. 3f:HT1 was stably expressed in 2D10 cells (D-HT cells). Cell lysates were submitted to SDS-PAGE and WB using anti-Flag and anti-GAPDH Abs (lower panel). FACS analysis of GFP positive cells showed that HIV reactivation by PMA, SAHA or JQ1 was significantly impaired in D-HT cells when compared to 2D10 cells, as determined by a student t-test. *** represent differences with p < E-03 (p = 4.75E-05, p = 4.35E-05 and p = 7.62E-05 respectively). Error bars represent standard deviation from triplicate FACS analysis. B. Stable expression of HT1 inhibits HIV reactivation from J-Lat 9.2 cells. 3f:HT1 was stably expressed in J-Lat 9.2 cells (L-HT cells). Cell lysates were submitted to SDS-PAGE and WB using anti-Flag and anti-GAPDH Abs (lower panel). FACS analysis of GFP positive cells showed that HIV reactivation by PMA was significantly impaired in L-HT cells when compared to J-Lat 9.2 cells, as determined by a student t-test. *** represent differences with p < E-03 (p = 5.62E-05). Error bars represent standard deviation from triplicate FACS analysis.

Fig 5. Stable expression of HT1 inhibits HIV replication.

A. Stable expression of HT1 inhibits HIV-1 replication in C-HT, MO-HT, and MT-HT cells. 1E+06 cells (HT1-expressing CEM-HT, MOLT4-HT, MT4-HT and respective controls CEM, MOLT4 and MT4 cells) were challenged by HIV-1 NL43 infection. Virus production was assessed by Gag p24 ELISA in supernatants when cells were passaged at days 0, 2 and 4, and plotted. Error bars represent standard deviation from triplicate experiments. B. Stable expression of HT1 doesn’t inhibit early steps of HIV-1 infection in C-HT, MO-HT, and MT-HT cells. Single-round infection assays were performed in the same conditions as in Fig 5A above, but using replication-defective HXB2 Env-pseudotyped HIV-1 NL43ΔenvLuc. The relative amount of HIV DNA integrated in the cells’ genomic DNA was assessed after 24h by qPCR. Error bars represent standard deviation from triplicate experiments.

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HEXIM1-Tat chimera inhibits HIV-1 replication - PubMed (original) (raw)