DENV as one of the most widespread vector borne viruses

DENV, as one of the most widespread vector-borne viruses, infects millions of people each year worldwide. However, no clinically approved drugs are yet available against DENV. There is an urgent need to develop effective anti-DENV drugs for such treatment. In this study we tested IFN-α2a, chloroquine, and mycophenolic raltegravir potassium for their ability to inhibit DENV infection in our in vitro system. IFN, as a well-established antiviral cytokine, can block several RNA and DNA viruses at different stages from viral entry to release. Both chloroquine and mycophenolic acid have been shown to inhibit replication of DENV in the laboratory tests (Farias et al., 2013; Diamond et al., 2002). Consistent with previous studies, all of these inhibitors reduced DENV infection in HLCs at various levels, suggesting that our in vitro system may have potential utility for anti-DENV drug screening efforts.
Experimental Procedures
Author Contributions
Acknowledgments We would like to thank Dr. Charles Rice for Huh7.5 cells and Dr. Qianjun Li for DENV2. We thank Brian Washburn, Christy Hammack, and Sarah Ogden for technical assistance, and members of the Tang laboratory for helpful comments. This study was supported by NIH/NIAID grant R21 AI119530 to H.T.
Introduction The RASopathies are developmental disorders caused by mutations in the RAS/MAPK pathway, characterized by pleomorphic developmental defects including facial dysmorphism, short stature, neurocognitive delay, and cardiac defects. One of the commonest cardiac manifestations is hypertrophic cardiomyopathy (HCM) (Tartaglia and Gelb, 2010). HCM is defined as thickening of the myocardium that occurs in the absence of an underlying insult, usually resulting from mutations in various genes encoding sarcomeric components. Histologically, there is cardiomyocyte (CM) enlargement and increased fibrosis. HCM is molecularly characterized by upregulation of a fetal gene program including increased expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), often with dysregulated calcium handling (Konno et al., 2010). About 70% of patients with HCM develop obstruction, while other complications include arrhythmias, heart failure, and sudden cardiac death (Harris et al., 2006). The role of RAS/MAPK signaling in cardiac hypertrophy remains unclear. Cardiac-restricted overexpression of Mek1 caused compensated hypertrophy with increased cardiac function (Bueno et al., 2000). In addition, Erk1−/−Erk2+/− mice were not protected from pressure overload or agonist stimulation (Purcell et al., 2007). However, oncogenic Hras over-expression led to pathological hypertrophy with fibrosis and calcium-handling defects (Hunter et al., 1995; Zheng et al., 2004), and dominant-negative Raf1 overexpression prevented pressure overload-induced cardiac hypertrophy (Harris et al., 2004). Some suggest that the pathological effects of RAS and RAF signaling may not be exclusively mediated by downstream MAPK signaling but rather by cross-activation of other pathways (Heineke and Molkentin, 2006). Mice expressing the RASopathy Raf1 allele developed HCM, rescued by MEK inhibition (Wu et al., 2011). However, mice with activating BRAF mutations did not exhibit pathological cardiac remodeling (Andreadi et al., 2012; Urosevic et al., 2011). Thus, the pathogenesis of HCM in cardio-facio-cutaneous syndrome (CFCS), whereby 75% of cases have germline BRAF mutations (Rodriguez-Viciana et al., 2006) and 40% develop HCM (Armour and Allanson, 2008), is unclear. To study this, we generated an hiPSC model of CFCS and developed a method to examine hiPSC-derived cell type-specific phenotypes and cellular interactions underpinning HCM by cell sorting based on SIRPα and CD90 expression. We show that purified CMs derived from hiPSCs harboring the CFC-causing T599R or Q257R BRAF mutations display cellular hypertrophy and intrinsic calcium-handling defects. In addition, fibroblast-like cells (FLCs) derived from BRAF-mutant hiPSCs exhibit pro-fibrotic behavior and modulate the hypertrophic phenotype through paracrine transforming growth factor β (TGFβ) signaling. Both TGFβ and RAS/MAPK inhibition rescue the hypertrophic phenotype.