Characterization of novel inhibitors of HIV-1 replication that function via alteration of viral RNA processing and rev function

Expression of the complete HIV-1 genome depends on the appropriate processing of viral RNA. Altering the balance of viral RNA processing impairs replication of the virus. In this report, we characterize two small molecule modulators of HIV-1 RNA processing, 8-azaguanine and 2-(2-(5-nitro-2-thienyl)vinyl)quinoline (5350150), which function by distinct mechanisms to suppress viral gene expression. Although only 8-Azaguanine dramatically decreased accumulation of HIV-1 unspliced and singly spliced RNAs and altered splice site usage, both compounds blocked Gag and Env expression without affecting production of Tat (p16) and Rev regulatory proteins. Subsequent analyses suggest that these compounds affect Rev-mediated RNA transport by different mechanisms. Both compounds induced cytoplasmic accumulation of Rev, suggesting that they function, in part, by impairing Rev function. This conclusion is supported by the determination that both drugs block the nuclear export of genomic HIV-1 RNA to the cytoplasm. Testing confirmed that these compounds suppress HIV-1 expression in T cells at doses below those previously used in humans for tumour chemotherapy. Together, our observations demonstrate that small molecules can be used to inhibit HIV-1 replication by altering another avenue of viral RNA processing, offering the potential for the development of novel therapeutics for controlling this disease

Differential effect of CLK SR Kinases on HIV-1 gene expression: potential novel targets for therapy

<p>Abstract</p> <p>Background</p> <p>RNA processing plays a critical role in the replication of HIV-1, regulated in part through the action of host SR proteins. To explore the impact of modulating SR protein activity on virus replication, the effect of increasing or inhibiting the activity of the Cdc2-like kinase (CLK) family of SR protein kinases on HIV-1 expression and RNA processing was examined.</p> <p>Results</p> <p>Despite their high homology, increasing individual CLK expression had distinct effects on HIV-1, CLK1 enhancing Gag production while CLK2 inhibited the virus. Parallel studies on the anti-HIV-1 activity of CLK inhibitors revealed a similar discrepant effect on HIV-1 expression. TG003, an inhibitor of CLK1, 2 and 4, had no effect on viral Gag synthesis while chlorhexidine, a CLK2, 3 and 4 inhibitor, blocked virus production. Chlorhexidine treatment altered viral RNA processing, decreasing levels of unspliced and single spliced viral RNAs, and reduced Rev accumulation. Subsequent experiments in the context of HIV-1 replication in PBMCs confirmed the capacity of chlorhexidine to suppress virus replication.</p> <p>Conclusions</p> <p>Together, these findings establish that HIV-1 RNA processing can be targeted to suppress virus replication as demonstrated by manipulating individual CLK function and identified chlorhexidine as a lead compound in the development of novel anti-viral therapies.</p

Identification of Novel Compounds That Inhibit HIV-1 Gene Expression by Targeting Viral RNA Processing

Novel strategies targeting different stages of the HIV lifecycle are vital for continued success in combating viral infection. Since HIV gene expression is dependent upon controlled splicing of the viral transcript, small molecule modulators of RNA processing hold tremendous promise as novel drugs. To this end, we screened splicing modulators for their effect on HIV-1 gene expression. We identified four compounds, 191, 791, 833 and 892, that strongly suppressed accumulation of HIV-1 incompletely spliced RNA and expression of viral structural/regulatory proteins. Furthermore, compound treatment had limited effects on alternative splicing of host RNA splicing events. Subsequent studies confirmed anti-HIV activity of two compounds in the context of peripheral blood mononuclear cells. The distinct effects of these compounds from previously characterized HIV-1 RNA processing inhibitors validate targeting this stage of the virus lifecycle. Elucidating the mechanism by which these compounds alter HIV-1 gene expression holds key insights for novel therapeutic strategies.M.Sc.2016-11-20 00:00:0

Differential effect of CLK SR Kinases on HIV-1 gene expression: potential novel targets for therapy

Abstract Background RNA processing plays a critical role in the replication of HIV-1, regulated in part through the action of host SR proteins. To explore the impact of modulating SR protein activity on virus replication, the effect of increasing or inhibiting the activity of the Cdc2-like kinase (CLK) family of SR protein kinases on HIV-1 expression and RNA processing was examined. Results Despite their high homology, increasing individual CLK expression had distinct effects on HIV-1, CLK1 enhancing Gag production while CLK2 inhibited the virus. Parallel studies on the anti-HIV-1 activity of CLK inhibitors revealed a similar discrepant effect on HIV-1 expression. TG003, an inhibitor of CLK1, 2 and 4, had no effect on viral Gag synthesis while chlorhexidine, a CLK2, 3 and 4 inhibitor, blocked virus production. Chlorhexidine treatment altered viral RNA processing, decreasing levels of unspliced and single spliced viral RNAs, and reduced Rev accumulation. Subsequent experiments in the context of HIV-1 replication in PBMCs confirmed the capacity of chlorhexidine to suppress virus replication. Conclusions Together, these findings establish that HIV-1 RNA processing can be targeted to suppress virus replication as demonstrated by manipulating individual CLK function and identified chlorhexidine as a lead compound in the development of novel anti-viral therapies

Digoxin Suppresses HIV-1 Replication by Altering Viral RNA Processing

<div><p>To develop new approaches to control HIV-1 replication, we examined the capacity of recently described small molecular modulators of RNA splicing for their effects on viral RNA metabolism. Of the drugs tested, digoxin was found to induce a dramatic inhibition of HIV-1 structural protein synthesis, a response due, in part, to reduced accumulation of the corresponding viral mRNAs. In addition, digoxin altered viral RNA splice site use, resulting in loss of the essential viral factor Rev. Digoxin induced changes in activity of the CLK family of SR protein kinases and modification of several SR proteins, including SRp20 and Tra2β, which could account for the effects observed. Consistent with this hypothesis, overexpression of SRp20 elicited changes in HIV-1 RNA processing similar to those observed with digoxin. Importantly, digoxin was also highly active against clinical strains of HIV-1 <i>in vitro</i>, validating this novel approach to treatment of this infection.</p> </div

Digoxin inhibits HIV-1 replication in PBMCs.

<p>(A–B) PHA/IL-2-activated human PBMCs were infected with R5 BaL strain of HIV-1 at a MOI of 10⁻². Following infection, cells were incubated in the presence of indicated doses of digoxin or DMSO (control). Equal concentrations of DMSO were present in each treatment. (A) After 8–10 days, media was harvested for analysis of HIV-1 Gag protein expression by p24^CA ELISA. Peak levels of Gag expression averaged ∼800 pg/ml at ∼8 days post-infection. ELISA results were averaged from ≥4 experiments and displayed as a fraction relative to DMSO (+) control, error bars are SEM, and statistical significant changes of treatments from DMSO control are indicated by asterisks as detailed in “<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003241#s4" target="_blank">Materials & Methods</a>”. (B) Cells were assessed following digoxin treatment for cell viability by Trypan blue exclusion (TB, black diamonds) or XTT assay (grey circles) as indicated. Shown is data averaged from ≥5 and ≥3 experiments, respectively, and displayed as a fraction relative to DMSO (+) control, error bars are SEM, and statistical significance indicated by asterisks as mentioned above. (C–F) CD8⁺-depleted PBMCs from chronically HIV-1-infected patients were activated by treatment with anti-CD3 and anti-CD28 antibodies to induce virus growth in cell culture. Cells were treated with either (C, D) digoxin or (E, F) the NRTI, 3TC. Media was harvested at multiple times points to assess virus replication by p24^CA ELISA of Gag. Shown (C, E) are the viral growth curves from 20 days of cell culture and (D, F) the dose response effects of the indicated drugs after only 14 days of culture. Effect of drugs on cell viability (Cell Viab., grey circles) was also tested in parallel by XTT assay. Note: cell viability data for (C, E) are shown on the y-axis adjacent to Gag levels while (D, F) is displayed in a similar manner but on the same y-axis as Gag expression. Inhibition and viability results shown (C–F) are derived from assays performed on three different patients, expressed relative to DMSO-treated cells, error bars are SEM, and statistical significance indicated by asterisks as described above.</p

Overexpression of SRp20, Tra2β1, or Tra2β3 suppress HIV-1 expression.

<p>(A) Diagram of SRp20, Tra2β1, and Tra2β3 protein domain structure. (B) Cells were transfected with the vectors CMVmyc (mock), CMVmyc SRp20 (SRp20), CMVmyc Tra2β1 (Tra2β1), or CMVmyc Tra2β3 (Tra2β3). Forty-eight hours post-transfection, cells were harvested, lysed in RIPA buffer, and proteins analyzed by western blot with anti-myc antibody followed by anti-tubulin (Tubulin) antibody to indicate loading. (C) HeLa rtTA-HIV-Δ<i>Mls</i> cells were transfected with (+) (or without, −) CMVtTApA (tTA) to activate the endogenous HIV-1 provirus along with CMV PLAP and either CMVmyc, CMVmyc SRp20, CMVmyc Tra2β1, or CMVmyc Tra2β3. Forty-eight hours post transfection, cell media was harvested and assayed by p24^CA ELISA for expression of HIV-1 Gag (black) and production of SEAP (white). Results are averaged from 6 independent assays, error bars are SEM, and asterisks indicate values deemed significant from CMVmyc (+tTA) as detailed in “<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003241#s4" target="_blank">Materials & Methods</a>”. (D) In parallel, total RNA was extracted from transfected cells and the abundance of individual viral RNA (unspliced, US; singly spliced, SS; multiply spliced, MS) were determined by qRT-PCR. Shown are results averaged from 6 independent assays, error bars are SEM, and asterisks indicate significance as noted above. (E, F) MS HIV-1 RNAs from transfected cells were amplified by RT-PCR as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003241#ppat-1003241-g004" target="_blank">Fig. 4</a>. Amplicons were fractionated on urea-PAGE gels and a representative gel is shown (E). Relative abundance of individual MS viral RNA species were quantitated and graphed (F). Data was averaged from >5 independent assays, errors bars are SEM, and asterisks indicate statistical significance as described above.</p

Digoxin blocks cytoplasmic accumulation of HIV-1 US RNA and its effect is partially reversed by expression of Rev <i>in trans</i>.

<p>(A) To assess the effect of digoxin treatment on viral RNA transport, in situ hybridization of US RNA was performed on HeLa rtTA-HIV-Δ<i>Mls</i> cells treated with digoxin, 3TC, or DMSO solvent. After 4 h, viral expression was left uninduced (−) or induced by addition of Dox (+). After 24 h, cells were fixed and incubated with labeled oligonucleotides specific to HIV-1 US RNA (US RNA). After washing to remove unbound probe, cells were stained with DAPI to allow detection of nuclei and images captured at 630× magnification. Shown is a representative sample of the results observed from ≥4 independent experiments. (B) To determine whether Rev expression <i>in trans</i> could alleviate the inhibition of Gag protein synthesis by digoxin, cells were transfected with vectors expressing dsRed or a dsRed-Rev fusion protein. At 24 h post-transfection, cells were treated with digoxin for 4 h then Dox was added to induce provirus expression. At 24 h post-doxycycline addition, cells were fixed, DAPI stained, and viewed for co-expression of dsRed (Cy3) and Gag signal (FITC). Shown is a representative sample of the results obtained. Note: intense foci in the nuclei seen in the FITC channel in cells transfected with dsRed-Rev is due to bleed through from the Cy3 channel. To assess the extent of reversing the effects of digoxin by expressing Rev in trans (C), >100 dsRed+ cells were examined in each experiment and the frequency of dsRed+ cells also having Gag+ expression was determined for each condition and displayed as “% of cells rescued”. Error bars of the data are SEM and significant changes from dsRed indicated by asterisk as detailed in “<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003241#s4" target="_blank">Materials & Methods</a>”.</p