Identification of a Small Molecule That Compromises the Structural Integrity of Viroplasms and Rotavirus Double-Layered Particles

Abstract

Despite the availability of two attenuated vaccines, rotavirus (RV) gastroenteritis remains an important cause of mortality among children in developing countries, causing about 215,000 infant deaths annually. Currently, there are no specific antiviral therapies available. RV is a nonenveloped virus with a segmented double-stranded RNA genome. Viral genome replication and assembly of transcriptionally active double-layered particles (DLPs) take place in cytoplasmic viral structures called viroplasms. In this study, we describe strong impairment of the early stages of RV replication induced by a small molecule known as an RNA polymerase III inhibitor, ML-60218 (ML). This compound was found to disrupt already assembled viroplasms and to hamper the formation of new ones without the need for de novo transcription of cellular RNAs. This phenotype was correlated with a reduction in accumulated viral proteins and newly made viral genome segments, disappearance of the hyperphosphorylated isoforms of the viroplasm-resident protein NSP5, and inhibition of infectious progeny virus production. In in vitro transcription assays with purified DLPs, ML showed dose-dependent inhibitory activity, indicating the viral nature of its target. ML was found to interfere with the formation of higher-order structures of VP6, the protein forming the DLP outer layer, without compromising its ability to trimerize. Electron microscopy of ML-treated DLPs showed dose-dependent structural damage. Our data suggest that interactions between VP6 trimers are essential, not only for DLP stability, but also for the structural integrity of viroplasms in infected cells.IMPORTANCE Rotavirus gastroenteritis is responsible for a large number of infant deaths in developing countries. Unfortunately, in the countries where effective vaccines are urgently needed, the efficacy of the available vaccines is particularly low. Therefore, the development of antivirals is an important goal, as they might complement the available vaccines or represent an alternative option. Moreover, they may be decisive in fighting the acute phase of infection. This work describes the inhibitory effect on rotavirus replication of a small molecule initially reported as an RNA polymerase III inhibitor. The molecule is the first chemical compound identified that is able to disrupt viroplasms, the viral replication machinery, and to compromise the stability of DLPs by targeting the viral protein VP6. This molecule thus represents a starting point in the development of more potent and less cytotoxic compounds against rotavirus infection.

Keywords: DLP; ML-60218; RNA polymerase III; VP6; antivirals; drug; inhibitor; rotavirus; viroplasms.

FIG 1

ML effect on RV replication. (A to E) Western blot and confocal immunofluorescence analyses with the indicated antibodies of RV-infected cells (MOI, 25 VFU/cell) treated with ML at 10 μM, unless otherwise indicated, or with DMSO (D) for the indicated times. Scale bars, 10 μm. TUB, tubulin. (F) Time course of viral progeny yield of OSU-infected (MOI, 25 VFU/cell) MA104 cells treated with 10 μM ML, added at 2 hpi. The data are presented as averages ± standard deviations of the results of three independent experiments. ***, P < 0.001 (t test). (G) Genome segment analysis of blotted total RNA extracted from noninfected (NI) and OSU-infected (25 VFU/cell) MA104 cells treated with ML (10 μM) or DMSO from 1 to 8 hpi and revealed with an anti-dsRNA antibody. (H) Viability of noninfected or OSU-infected (MOI, 25 VFU/cell) MA104 cells determined by cytofluorometry of propidium iodide-stained cells following treatment at 2 hpi with or without 10 μM ML for up to 12 hpi. The data are presented as averages ± standard deviations of the results of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (t test).

FIG 2

Electron microscopy of RV-infected cells treated with ML. High-definition electron microscopy of noninfected (NI) and RV-infected (OSU; MOI, 100 VFU/ml) MA104 cells untreated (DMSO) or treated with ML (20 μM) from 1 hpi. At 6 hpi, the cells were fixed with glutaraldehyde and processed for transmission electron microscopy. V, viroplasms; Nu, nucleus, ER, endoplasmic reticulum; Gg, Golgi complex; Vc, vacuoles; P_h, phagosomes; CM, cell membrane; the thin arrows indicate the endoplasmic reticulum membrane surrounding viroplasms; the large arrowheads indicate viral particles.

FIG 3

NSP5 dephosphorylation caused by ML-mediated viroplasm disruption. Shown are Western blot and confocal immunofluorescence analyses with the indicated antibodies of OSU-infected (25 VFU/cell) MA104 cells treated with 10 μM ML and/or 0.5 μM okadaic acid (OA) or DMSO for the indicated times. Scale bars, 5 μm.

FIG 4

ML antiviral activity is independent of cellular transcription and protein synthesis. (A) Western blot and confocal immunofluorescence analyses with the indicated antibodies of OSU-infected MA104 cells (25 VFU/cell) fed with EU and treated with 10 μM ML and/or 10 μg/ml actinomycin D (Act D) or DMSO for the indicated times. EU-labeled, newly synthesized RNAs were visualized by reaction with an Alexa-488-conjugated azide (green). Scale bars, 5 μm. (B) Western blot and confocal immunofluorescence analyses with the indicated antibodies of OSU-infected (MOI, 25 VFU/cell) MA104 cells treated with 10 μM ML and/or 10 μg/ml CHX or DMSO for the indicated times. Scale bars, 10 μm.

FIG 5

ML-mediated impairment of DLP stability. (A) Transcriptional activity of purified DLPs. The plot shows the dose-dependent decrease of transcripts produced by SA11 or OSU DLPs incubated in the presence of the indicated concentrations of ML. The small molecule 7749832 (ChemBridge Corp.) at 200 μM was used as an irrelevant compound (irr.). The data represent the means ± standard deviations of the results of at least three independent experiments. **, P < 0.01; ***, P < 0.001; ns, P > 0.01 (t test). (B and C) DLP morphology analyzed by electron microscopy. Purified DLPs were incubated for 4 h with the indicated concentrations of ML or DMSO. The arrows indicate damaged DLPs with an irregular shape and partially open. Quantification of damaged DLPs is shown in panel C. The data are presented as averages ± standard errors of the mean (SEM). ***, P < 0.001 (t test); n > 100.

FIG 6

Effect of ML on RV VP6. (A) VP6-VP2 interaction. Shown is Western blot analysis with anti-VP2 and anti-VP6 antibodies of immunoprecipitates (IP) obtained with anti-VP6 MAb RV138 from extracts of MA104 cells transfected with VP6 and VP2 and treated for 5 h with 10 μM ML or DMSO. The inhibitor (200 μM) was maintained during cell lysis and incubation with the precipitating antibody. The numbers on the left are kilodaltons. (B) VP6 trimer stability. (Left) Western blot analysis with anti-VP6 antibody of nonboiled extracts from MA104 cells infected with a recombinant vaccinia virus expressing VP6 (VVVP6) and treated with 10 μM ML or DMSO from 1 to 7 hpi. (Right) Western blot analysis of nonboiled extracts from cells infected with OSU (MOI, 25 VFU/cell) and treated with 10 μM ML or DMSO from 1 to 5 hpi. The numbers on the right are kilodaltons. (C) Confocal immunofluorescence (IF) analysis with the anti-VP6 MAb 4B2D2 of MA104 cells overexpressing VP6 (infected with VVVP6) and treated with 10 μM ML or DMSO from 1 to 7 hpi. The arrow indicates a VP6 higher-order structure observed in the absence of other RV proteins. Scale bars, 5 μm. (D) Representative images of VP6 tubes and spheres visualized by negative-staining electron microscopy after treatment with 25 μM ML for 4 h at 37°C. (E) Interaction of VP6 with ML evaluated by nanoscale thermophoresis. The fraction of Cys- or Lys-labeled VP6 bound to ML was plotted against increasing concentrations of the inhibitor. The data were fitted with two state equations, and an EC₅₀ of 294 ± 62 μM was calculated as the average of the results of three independent measurements. The error bars indicate standard deviations.

FIG 7

ML effect on VLS. Confocal immunofluorescence assay of VLS (A and C) and Western blot analysis (B) with the indicated antibodies of MA104 cells transfected with NSP5, NSP2, VP2, and VP6, as indicated. (A) NSP5 is shown in green and NSP2 or VP2 in red. (C) NSP5 is shown in red and VP6 in green. Cells were treated for 5 h with 10 μM ML or DMSO at 18 h posttransfection. Bars, 5 μm.

FIG 8

VP6 in RV-infected cells. Shown is confocal immunofluorescence of MA104 cells infected with either OSU or SA11 (MOI, 25 VFU/cell) and transfected with siRNAs specific for SA11 VP6 or OSU VP6 or with a nontargeting siRNA (siNT) (A) or treated with 10 μM ML or DMSO (B). At the indicated times postinfection, viroplasms were visualized with anti-NSP5 antibody (red) and VP6 with MAb 4B2D2 (green). Scale bars, 5 μm.