MK2a inhibitor CMPD1 abrogates chikungunya virus infection by modulating actin remodeling pathway

Abstract

Chikungunya virus (CHIKV) epidemics around the world have created public health concern with the unavailability of effective drugs and vaccines. This emphasizes the need for molecular understanding of host-virus interactions for developing effective targeted antivirals. Microarray analysis was carried out using CHIKV strain (Prototype and Indian) infected Vero cells and two host isozymes, MAPK activated protein kinase 2 (MK2) and MAPK activated protein kinase 3 (MK3) were selected for further analysis. The substrate spectrum of both enzymes is indistinguishable and covers proteins involved in cytokines production, endocytosis, reorganization of the cytoskeleton, cell migration, cell cycle control, chromatin remodeling and transcriptional regulation. Gene silencing and drug treatment were performed in vitro and in vivo to unravel the role of MK2/MK3 in CHIKV infection. Gene silencing of MK2 and MK3 abrogated around 58% CHIKV progeny release from the host cell and a MK2 activation inhibitor (CMPD1) treatment demonstrated 68% inhibition of viral infection suggesting a major role of MAPKAPKs during late CHIKV infection in vitro. Further, it was observed that the inhibition in viral infection is primarily due to the abrogation of lamellipodium formation through modulation of factors involved in the actin cytoskeleton remodeling pathway. Moreover, CHIKV-infected C57BL/6 mice demonstrated reduction in the viral copy number, lessened disease score and better survivability after CMPD1 treatment. In addition, reduction in expression of key pro-inflammatory mediators such as CXCL13, RAGE, FGF, MMP9 and increase in HGF (a CHIKV infection recovery marker) was observed indicating the effectiveness of the drug against CHIKV. Taken together it can be proposed that MK2 and MK3 are crucial host factors for CHIKV infection and can be considered as important target for developing effective anti-CHIKV strategies.

Fig 1. Differential host gene expressions for PS and IS strains of CHIKV in Vero cells.

(A) Hierarchical clustering showing the overall expression patterns of the modulated host genes by PS/IS strains of CHIKV during infection in mammalian cells. (B) Pie-chart depicting the distribution of the host genes in CHIKV-infected samples into different protein classes. (C) Pie-chart depicting the distribution of the modulated host genes into different cellular processes. (D and E) Venn diagram showing both commonly and differentially regulated host genes in CHIKV (PS/IS) infected Vero cells.

Fig 2. MK2 and MK3 gene silencing abrogates CHIKV progeny release without affecting viral protein synthesis.

(A and B) After 24 hrs post transfection with 60 pmol of MK2/3 siRNA (either separately/in combination), cells were super-infected (PS/IS MOI 0.1) and harvested at 18 hpi. Western blot showing the expression levels of different proteins (Left panel). Bar diagrams showing relative band intensities of different proteins (Right panel). GAPDH was used as control. (C and D) Bar diagram showing the viral titres after siRNA treatment for PS and IS strains, (n  =  3; p<0.05).

Fig 3. CMPD1, an MK2a inhibitor abrogates CHIKV infection in vitro.

(A) Vero cells were treated with different concentrations of CMPD1 (25, 50, 75 and 100 μM) for 24 h and MTT assay was performed. (B and C) Vero cells infected with CHIKV PS/IS at MOI 0.1 and drug treated. Bar graph showing viral titers in the presence of CMPD1 (25, 50 and 100 μM). (D) Dose response curve showing the IC₅₀ of CMPD1 against CHIKV. (E) Bar graph showing the viral titers estimated through plaque assay from the supernatants obtained from the time of addition experiment for CMPD1 (50μM) post CHIKV infection. (F and G) Bar graph showing intracellular and extracellular virus titers for samples harvested at 18hpi. DMSO was used as control. All the graphs depict the values of mean ± SD (*p< 0.05) of three independent experiments.

Fig 4. CMPD1 blocks the actin polymerization process modulated by CHIKV for its progeny release.

Vero cells were infected with the IS strain (0.1 MOI), 50μM of CMPD1 was added to the cells and incubated for 18 hpi. (A) Bright field images (20X magnification) showing the cytopathic effect after CHIKV infection with or without CMPD1 treatment (50μM). (B) Western blot analysis showing the expressions of nsP2, pMK2, MK3, Cofilin and p-Cofilin proteins. GAPDH served as the loading control. (C) Bar graphs showing the relative fold change in viral and host proteins expression with respect to DMSO control. (D) Confocal microscopy images showing the levels of E2 and phalloidin during CHIKV infection.

Fig 5. CMPD1 inhibits CHIKV infection in mice.

(A) Bar graph showing the viral copy numbers in CHIKV infected and CMPD1 treated mouse serum samples. (B) H and E staining of mouse tissue samples with CHIKV infection and in presence/absence of CMPD1(C) Array blot showing the expression of different cytokines after CHIKV infection in presence and absence of CMPD1. (D) Bar graph showing the relative band intensities of selected cytokines in mock, CHIKV infected and CMPD1 treated samples. (E) Survival curve showing the effect of CMPD1 in CHIKV infected mice.

Fig 6. Proposed model for CHIKV infection.

(A) During CHIKV infection, MK2/3 gets phosphorylated by P38 MAPK thereby exposing the Nuclear Export Signal (NES) of MK2. The phosphorylated forms of MK2/MK3 translocate to the cytoplasm and help in inactivating Cofilin through phosphorylation via LIMK-1 thereby promoting actin polymerization and lamellipodium formation. (B) Addition of CMPD1 blocks the phosphorylation of MK2, thereby blocking Cofilin phosphorylation and eventually inhibiting lamellipodium formation and CHIKV progeny release.