c-Src protein kinase inhibitors block assembly and maturation of dengue virus

Abstract

Dengue virus is a mosquito-borne flavivirus that represents an important emerging infectious disease and is an international health concern. Currently, there is no vaccine or effective antiviral therapy to prevent or to treat dengue virus infection. The slow progress in developing antiviral agents might be alleviated by the availability of efficient high-throughput anti-dengue virus screening assays. In this study, we report an immunofluorescence image-based assay suitable for identification of small molecule inhibitors of dengue virus infection and replication. Using this assay, we have discovered that inhibitors of the c-Src protein kinase exhibit a potent inhibitory effect on dengue virus (serotypes 1-4) and murine flavivirus Modoc. Mechanism of action studies demonstrated that the c-Src protein kinase inhibitor dasatinib prevents the assembly of dengue virions within the virus-induced membranous replication complex. These results demonstrate that this cell-based screen may provide a powerful means to identify new potential targets for anti-dengue drug development while simultaneously providing pharmacological probes to investigate dengue virus-host cell interactions at the biochemical level. Given the simplicity and excellent reproducibility of the assay, it should be useful in high-throughput screens of both small molecule and RNAi libraries when implemented on a robotic image-based high-throughput screen (HTS) platform. Given the reasonable clinical safety of inhibitors such as dasatinib and AZD0530, inhibitors of c-Src protein kinase may have the potential to become a new class of anti-dengue viral therapeutic agents.

Fig. 1.

Development of an image-based immunofluorescence assay for detection of DENV. (A) Detection of DENV infection of Vero cells using immunofluorescence staining for DENV envelope protein (Env) in the absence and presence of MPA. (B) Dosage-dependent inhibition of DENV 2 infection of cells treated with MPA.

Fig. 2.

Antiviral activity of dasatinib, an inhibitor of c-Src and Abl kinases, on DENV infection. (A) Dosage dependent inhibition of DENV2 infection was observed in C6/36, Vero, and Huh-7 cells. (B) Viral reduction immunofluorescence assays were performed for the indicated viruses to determine the dose-dependent anti-DENV activity of dasatinib.

Fig. 3.

siRNA Knockdown of c-Src protein levels inhibits DENV infection. Huh-7 cells were transfected with different concentrations of a c-Src-specific siRNA pool or control siRNA. (A) Knockdown of c-Src protein expression was confirmed by Western blot analysis by using a c-Src specific antibody. Lane 1, Control (siGlo RNA-300 nM); lane 2, c-Src specific siRNA pool–200 nM; lane 3, c-Src specific siRNA pool–300 nM. Detection of GAPDH was included as a well loading control. (B) DENV2 infection was reduced by >1.5-log units in cells transfected with 200 nM of the c-Src siRNA pool but was unchanged in the presence of 200 nM siGlo or 200 nM GAPDH SMARTpool negative control reagents.

Fig. 4.

Inhibition of viral spread in dasatinib-treated, DENV-infected cells. An immunofluorescence assay was conducted to detect the localization of the viral envelope (E) protein in DENV-infected Vero, Huh-7, and C6/36 cells that were treated with 2.5 μM of dasatinib or DMSO. Noninfected cells are indicated by arrowheads, and accumulation of viral E protein in the perinuclear region is indicated by arrows. Cell nuclei are stained blue with DAPI.

Fig. 5.

Inhibition of c-Src protein kinase activity prevents the assembly of DENV within the ER lumen. DENV2-infected Vero cells treated with either DMSO or 2.5 μM of dasatinib (A and B, respectively) and mock-infected cells treated with DMSO (C) were processed for electron microscopy at 4 days postinfection. (A) Representative DENV particles localized within the ER lumen are indicated by arrows. (B) Extensive proliferation of virus-induced membranes is indicated by arrowheads and nucleocapsid-like particles are noted by the arrows. No virus particles are observed within the ER lumen and membranous structures. (C) Typical ultrastructural morphology of noninfected Vero cells. (Scale bars: 200 nm for A–C.)