Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays

Abstract

Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC₅₀ values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening.

Figure 1. Construction and characterization of a stable BHK cell line carrying CHIKV-replicon.

A) Schematic representation of the used CHIKV replicons (numbers and symbols are explained in the text). B) The process leading to selection of non-cytotoxic (NCT) CHIKV replicons, identification of mutations associated with the NCT phenotype and confirmation of their phenotypes. C) Phenotype of BHK-CHIKV-NCT cells; green fluorescence is caused by EGFP expression. Arrow indicates a cell in the process of division. D) Immunofluorescence images of BHK-CHIKV-NCT cells stained with anti-dsRNA (top), anti-SFV nsP3 (middle) and co-staining with anti-dsRNA and anti-SFV nsP3 (bottom). A representative optical slice from the middle of the cell is shown. Scale bar is 10 µm.

Figure 2. Effects of adaptive mutations on the CHIKV replicon.

A) Effects of the PG and NCT mutations on the accumulation of positive-strand replicon and corresponding sgRNAs in cells transfected with in vitro transcripts of CHIKV-LR, CHIKV-PG, CHIKV-NCT and their variants containing the Rluc marker in the nsP3 region. Total RNAs extracted from transfected cells at 16 h post-transfection; 10 µg aliquot from each sample was separated by electrophoresis in formaldehyde gel and analyzed by Northern blotting. The constructs are shown at the top; positions of replicon and sgRNAs are indicated with arrows. “Mock” indicates RNAs from mock-transfected control cells. B) Immunofluorescence analysis of nsP2 localization in cells transfected with in vitro transcripts of CHIKV-LR or CHIKV-PG at 8 h post-infection or with in vitro transcripts of CHIKV-NCT at 16 h post-infection. Cells were fixed and stained with anti-nsP2 and DAPI; EGFP was detected by fluorescence. Merge indicates co-staining of DAPI and nsP2.

Figure 3. Virus entry assays with SFVts9-Rluc.

A) The temperature-sensitive phenotype of SFVts9-Rluc as measured by Rluc activity of infected cell lysates at 1 h, 2 h and 3 h post-infection at 39°C. B) The effect of chloroquine on Rluc signals of SFVts9-Rluc at 39°C. C) Effects of 5,7-dihydroxyflavones and 10H-phenothiazines on the accumulation of Rluc in cells infected with SFVts9-Rluc at 39°C. Selected examples of results of hit compounds in SFV entry inhibition assay. The cell cultures were treated with 100 µM compounds and Rluc levels were determined 1 h post-infection. All results represent the average of three replicates.