The propagation of the hepatitis C virus (HCV) is a complex process that requires both host and viral proteins. To facilitate identification of host cell factors that are required for HCV replication, we screened a panel of small interference RNAs that preferentially target human protein kinases using an HCV replicon expressing the firefly luciferase gene as a genetic reporter. Small interference RNAs specific for three human kinases, Csk, Jak1, and Vrk1, were identified that reproducibly reduce viral RNA and viral protein levels in HCV replicon-bearing cells. Treatment of replicon cells with a small molecule inhibitor of Csk also resulted in a significant reduction in HCV RNA and proteins, further supporting a role for Csk in HCV replication. The effects of siRNAs targeting eight kinases known to be negatively regulated by Csk were then examined; knock down of one of these kinases, Fyn, resulted in up-regulation of the HCV replicon, suggesting that Csk mediates its effect on HCV replication through Fyn. This conclusion was further corroborated by demonstration that replicon cells treated with Csk inhibitor contained lower levels of the phosphorylated form of Fyn than control cells. Hepatitis C virus (HCV)2 has emerged as a major cause of human liver disease, with ϳ3% of the world population persistently infected with the virus and more than 1 million new cases of infection reported annually (1). In ϳ70% of the cases, HCV escapes the immune system and establishes a chronic infection. In the long term, these chronic carriers are at risk of developing life-threatening liver disease, including hepato-cellular carcinoma (1). HCV is an enveloped virus that belongs to the Hepacivirus genus in the Flaviviridae family. Its genome consists of RNA of positive polarity ϳ9.6 kb in length that contains a large open reading frame. Translated polyprotein is processed by cellular and viral proteases into at least 10 individual structural and nonstructural (NS) proteins. NS proteins are sufficient to support viral RNA replication and include the metalloprotease NS2, serine protease/helicase NS3, NS3 protease cofactor NS4A, RNA-dependent polymerase NS5B, and two other proteins with poorly characterized function, NS4B and NS5A. According to the current model, virus replication occurs within a complex that comprises viral RNA and NS proteins and is associated with the host endoplasmic reticulum (2, 3).Current therapy for hepatitis C involves treatment with a combination of interferon-␣ and ribavirin. However, this regimen is effective only in half of patients, often poorly tolerated, and unsuitable for certain patient populations (4). Thus, there is an intense effort to develop new, better treatments, mostly by targeting viral enzymes (5). A complementary approach is to inhibit nonessential host cell proteins that are required for the viral life cycle. Several such host cell factors have been identified to date and represent potential candidates for this strategy. For example, inhibition of geranylgeranylation by lovastatin, a smal...

Diabetic nephropathy is associated with interstitial macrophage infiltrates, but their contribution to disease progression is unclear. We addressed this question by blockade of chemokine receptor (CCR)1 because CCR1 mediates the macrophage recruitment to the renal interstitium. In fact, when CCR1 was blocked with BL5923, a novel orally available CCR1 antagonist, the interstitial recruitment of ex vivo labeled macrophages was markedly decreased in uninephrectomized male db/db mice with advanced diabetic nephropathy. Likewise, BL5923 (60 mg/kg, twice a day) orally administered from months 5 to 6 of life reduced the numbers of interstitial macrophages in uninephrectomized db/db mice. This was associated with reduced numbers of Ki-67 proliferating tubular epithelial and interstitial cells, tubular atrophy, and interstitial fibrosis in uninephrectomized db/db mice. Glomerular pathology and proteinuria were not affected by the CCR1 antagonist. BL5923 reduced renal mRNA expression of Ccl2, Ccr1, Ccr2, Ccr5, transforming growth factor-beta1, and collagen I-alpha1 when compared with untreated uninephrectomized male db/db mice of the same age. Thus, we identified a previously unrecognized role for interstitial macrophages for tubulointerstitial injury, loss of peritubular microvasculature, interstitial inflammation, and fibrosis in type 2 diabetic db/db mice. These data identify oral treatment with the CCR1 antagonist BL5923 as a potential therapy for late-stage diabetic nephropathy.

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Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is essential for immune responses triggered by antigen receptors but the contribution of its paracaspase activity is not fully understood. Here, we studied how MALT1 proteolytic function regulates T-cell activation and fate after engagement of the T-cell receptor pathway. We show that MLT-827, a potent and selective MALT1 paracaspase inhibitor, does not prevent the initial phase of T-cell activation, in contrast to the pan-protein kinase C inhibitor AEB071. However, MLT-827 strongly impacted cell expansion after activation. We demonstrate this is the consequence of profound inhibition of IL-2 production as well as reduced expression of the IL-2 receptor alpha subunit (CD25), resulting from defective canonical NF-jB activation and accelerated mRNA turnover mechanisms. Accordingly, MLT-827 revealed a unique transcriptional fingerprint of MALT1 protease activity, providing evidence for broad control of T-cell signaling pathways. Altogether, this first report with a potent and selective inhibitor elucidates how MALT1 paracaspase activity integrates several T-cell activation pathways and indirectly controls gammachain receptor dependent survival, to impact on T-cell expansion.

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