Kheir MM, Wang Y, Hua L, Hu J, Li L, Lei F, Du L. activated the major mitogen-activated protein kinase (MAPK) signaling pathways extracellular signal-related kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK). Upon treatment with berberine, this virus-induced MAPK activation was markedly reduced. Subsequent analyses with specific inhibitors of these kinases indicated that the ERK and JNK signaling cascades are important for the generation of progeny virions. In contrast to specific MAPK inhibitors, berberine lowered virus-induced activation of all major MAPK pathways and resulted in a stronger reduction in viral titers. Further, we assessed the efficacy of berberine in a mouse model and measured a significant reduction of CHIKV-induced inflammatory disease. In summary, we demonstrate the efficacy of berberine as a drug against CHIKV and highlight the importance of the MAPK signaling pathways in the alphavirus infectious cycle. IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne virus that causes severe and persistent muscle and joint pain and has recently spread to the Americas. No licensed drug exists to counter this 3-Methylcytidine virus. In this study, 3-Methylcytidine we report that the alkaloid berberine is antiviral against different CHIKV strains and in multiple human cell lines. We demonstrate that berberine collectively reduced the virus-induced activation of cellular mitogen-activated protein kinase signaling. The relevance of these signaling cascades in the viral life cycle was emphasized by specific inhibitors of these kinase pathways, which decreased the production of progeny virions. Berberine significantly reduced CHIKV-induced inflammatory disease in a mouse model, demonstrating efficacy of the drug mosquitoes and characterized by a sudden onset of febrile illness, nausea, headache, and most importantly, severe and persistent musculoskeletal pain (1). It reemerged in tropical Asia and Africa Rabbit Polyclonal to PEA-15 (phospho-Ser104) 1 decade ago and since 2013 has infected more than 1.5 million people in the Americas (2). So far, no licensed vaccine or antiviral treatment exists to counter this disease. Over the years, many research groups have focused on antiviral drug discovery against CHIKV, but very few of these potential antiviral candidates have been characterized in animal models to demonstrate their efficacy (3, 4). CHIKV is grouped in the genus (family (ONNV) and (SINV), which cause similar disease in humans, and low-pathogenic viruses such as (SFV). The CHIKV genome is a single-stranded positive-sense RNA, 12 kb in length, comprising two open reading frames that encode the nonstructural and structural proteins of the virus, respectively. Upon infection, nonstructural proteins 1 to 4 3-Methylcytidine (nsP1 to nsP4) are translated as a polyprotein from the genomic RNA, following which they induce membrane invaginations called spherules that act as active viral replication complexes. After the initial minus strand synthesis, the replication complex switches over to synthesis of full-length genomic 3-Methylcytidine RNA as well as the subgenomic RNA, from which the structural proteins (capsid and envelope proteins) are translated. The viral genomic RNA is then packaged into capsids and transported to the plasma membrane, the site of virus budding (5, 6). Virus infection typically manipulates cellular metabolism and signaling pathways to favor virus replication. CHIKV has been shown to modulate the prosurvival phosphatidylinositol-3 kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) axis (7), endoplasmic reticulum (ER) stress response pathways (8), and pattern recognition receptor-mediated innate immune pathways (9, 10). However, one major type of signaling network underexplored in the context of alphavirus infection is the mitogen-activated protein kinase (MAPK) signaling. The MAPKs are a group of signal-transducing proteins expressed ubiquitously in most mammalian cell types, primarily mediating the host cell response to diverse external stimuli. The basic structure of the typical MAPK signaling pathway follows a three-tiered cascade of activating kinases, generically termed MAPKKK (MAPK kinase kinase), MAPKK (MAPK kinase), and MAPK, to phosphorylate and activate a plethora of cytoplasmic and nuclear substrates, usually transcription factors with roles in cell growth, differentiation, proliferation, migration, and apoptosis (reviewed in references 11 and 12). The major MAPKs are extracellular signal-related kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK) (see schematic in Fig. 6A). Open in a separate window FIG 6 Effects of MAPK inhibitors on CHIKV. (A) Schematic diagram showing the three main groups of the MAPK signal transduction system: ERK, p38 MAPK, and JNK. Some of the main kinases at each stage are shown along with the inhibitors used in this study. (B) Experimental setup. BHK-21 cells were pretreated with berberine (5 M), U0126 (20 M), SB203580 (10 M), or SP600125 (40 M). DMSO (0.1%) was used as a solvent control. Following the pretreatment, cells.