Anti-HIV-1 Effect of the Fluoroquinolone Enoxacin and Modulation of Pro-Viral hsa-miR-132 Processing in CEM-SS Cells

Abstract

Background: Despite tremendous advances in antiretroviral therapy (ART) against HIV-1 infections, no cure or vaccination is available. Therefore, discovering novel therapeutic strategies remains an urgent need. In that sense, miRNAs and miRNA therapeutics have moved intensively into the focus of recent HIV-1-related investigations. A strong reciprocal interdependence has been demonstrated between HIV-1 infection and changes of the intrinsic cellular miRNA milieu. This interrelationship may direct potential alterations of the host cells' environment beneficial for the virus or its suppression of replication. Whether this tightly balanced and controlled battle can be exploited therapeutically remains to be further addressed. In this context, the fluoroquinolone antibiotic Enoxacin has been demonstrated as a potent modulator of miRNA processing. Here, we test the hypothesis that this applies also to selected HIV-1-related miRNAs.

Methods: We studied the effect of Enoxacin on HIV-1 replication coupled with miRNA qRT-PCR analysis of HIV-1-related miRNAs in CEM-SS and MT-4 T-cells. The effects of miRNA mimic transfections combined with Enoxacin treatment on HIV-1 replication were assessed. Finally, we employed an in vitro DICER1 cleavage assay to study the effects of Enoxacin on a pro-HIV-1 miRNA hsa-miR-132 processing.

Results: We established that Enoxacin, but not the structurally similar compound nalidixic acid, exhibits strong anti-HIV-1 effects in the T-cell line CEM-SS, but not MT-4. We provide experimental data that this effect of Enoxacin is partly attributed to the specific downregulation of mature hsa-miR-132-3p, but not other tested pro- or anti-HIV-1 miRNAs, which is likely due to affecting DICER1 processing.

Conclusions: Our findings show an anti-retroviral activity of Enoxacin at least in part by downregulation of hsa-miR-132-3p, which may be relevant for future antiviral therapeutic applications by modulation of the RNA interference pathway.

Keywords: DICER1 processing; Enoxacin; HIV-1; RNA interference; fluoroquinolones; hsa-miR-132; miRNA.

Figure 1

TaqMan qRT-PCR analysis of selected anti- and pro-HIV miRNAs in CEM-SS cells. Cells were treated for 4 and 7 days post-infection with 50 µM final concentration of (a) Enoxacin, (b) nalidixic acid relative to equivalent volume of DMSO control. * p < 0.05 or ** p < 0.01 two-tailed Student’s t-test. Error bars indicate ± 1 s.d.

Figure 2

Effect of Enoxacin and nalidixic acid on HIV-1 replication in (a) CEM-SS cells over two to ten days p.i. (red arrows indicate time points of qPCR in (c)) measured by p24-ELISA. (b) Fold inhibition of Enoxacin at 50 µM final concentration on HIV-1 replication compared to controls in CEM-SS and (c) MT-4 cells were spin-infected with HIV-1HXB2 at MOI 0.01 and monitored for p24 expression in cell culture supernatant. Untreated cells infected with HIV-1 served as positive, DMSO treatment as negative and no virus as background controls, respectively. (n = 4, technical duplicates, DMSO control = nominator, fold-inhibition = 1), ** p < 0.01, two-tailed Mann Whitney U test. Error bars indicate ± 1 s.d. (d) Pro-HIV-1 miR-132-3p relative expression mirrors the anti-HIV-1 effect of Enoxacin at days 4 and 7 but not control miR-223 (anti-HIV-1) or miR-23-a (T-cell miRNA) compared to DMSO by qRT-PCR. ** p < 0.01 two-tailed Student’s t-test. Error bars indicate ± 1 s.d., red arrows indicate time points of significance for p24-ELISA and corresponding qPCR.

Figure 3

Hsa-miR-132-3p enhances HIV-1 replication in CEM-SS cells. Cells were spin-infected with HIV-1HXB2 at MOI 0.01 and monitored for p24 expression in supernatant at days 0 and 10 post-infection (p.i.) reverse transfected with miR-mimic or scrambled control at 100 nM plus DMSO. DMSO/mock transfection served as negative and cells and virus alone as positive control. Grey arrow indicates time of transfection. n = 2, * p < 0.05 or ** p < 0.01, two-tailed Student’s t-test. Error bars indicate ± 1 s.d.

Figure 4

Hsa-miR-132-3p partially rescues Enoxacin-dependent anti-HIV-1 effect in CEM-SS cells. Cells were spin-infected with HIV-1HXB2 at MOI 0.01 and monitored for p24 expression in supernatant over 4 to 10 days post-infection (p.i.) in the presence of 50 µM final concentration of Enoxacin or respective equivalent volume of DMSO control as shown as fold HIV-1 inhibition by p24 ELISA. Untreated cells infected with HIV-1 served as positive control. Cells were reverse transfected in DMSO or Enoxacin treatment with 100 nM concentration of hsa-miR-132-3p mimic or scrambled control. DMSO and Enoxacin treatment with mock transfection served as additional controls. n = 2, two-tailed Mann-Whitney U-test, error bars indicate ± 1 s.d.

Figure 5

In vitro DICER1 cleavage assay in CEM-SS lysates (a) or recombinant DICER1 protein alone (b). miR-132 hairpin was 5′- (a) or 3′ (b) γ-phosphate labeled and incubated in 100 µM, 300 µM and 1 mM Enoxacin or DMSO control at 37 °C for between 0 and 60 min and PAGE-gel separated. Pre-miR and miR bands were densitometrically quantified and DICER1 processing effect was plotted as normalized ratio to DMSO (a) or % cleavage (b). Note: See full autoradiograms and other replicates Figure S5. (c) In vitro SHAPE-MaP reactivity scores plotted on RNAstructure prediction of pre-miR-132 in the presence of 150 and 300 µM Enoxacin and negative control (0 µM Enoxacin). Red triangles indicate DICER1 and blue triangles DROSHA cleavage sites. Light red sequences indicate significant structural rearrangements (marked by asterisks) determined by SHAPE scores of Enoxacin treatment vs. control.