Identification of Human Kinases Involved in Hepatitis C Virus Replication by Small Interference RNA Library Screening

Abstract

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...

Pan‐Src Family Kinase Inhibitors Replace Sox2 during the Direct Reprogramming of Somatic Cells

“…[12] For example, activation of c-Src signaling promotes ES cell differentiation. [13] Consistent with this observation we find that the activation of Src signaling in MEFs with JK239 [14] potently inhibits 4-factor reprogramming (Figure 2c). Together, our results suggest that SFK signaling is an important mediator of somatic cell reprogramming, where activation of the SFK pathway prevents reprogramming and inhibition allows for reprogramming in the absence of exogenous Sox2.…”

Host–virus interactions during hepatitis C virus infection: a complex and dynamic molecular biosystem

“…For example, siRNA-based screening has been successfully implemented to systematically knockdown genes within the host genome to discover the relative importance of host cell proteins and pathways. [146][147][148][149] For example, Supekova et al screened a panel of siRNAs that preferentially target human protein kinases against an HCV replicon expressing the firefly luciferase gene as a genetic reporter. 147 They identified three human kinases, Csk, Jak1, and Vrk1, that reduce viral RNA and protein levels.…”

“…[146][147][148][149] For example, Supekova et al screened a panel of siRNAs that preferentially target human protein kinases against an HCV replicon expressing the firefly luciferase gene as a genetic reporter. 147 They identified three human kinases, Csk, Jak1, and Vrk1, that reduce viral RNA and protein levels. They also demonstrated that a small molecule inhibitor of Csk, JK239 (Fig.…”

Dual regulation of hepatitis C viral RNA by cellular RNAi requires partitioning of Ago2 to lipid droplets and P-bodies

“…On the one hand, the HCV apparently evolved to utilize a highly abundant, well-conserved liver-specific miRNA-122 and key RNAi proteins, including Argonaute 1-4 and Dicer, for replication (Jopling et al 2005), translation of the viral genome (Henke et al 2008;Jangra et al 2010b), and infectious virus production (Randall et al 2007;Jangra et al 2010b). On the other hand, the cellular RNAi machinery is capable of potently suppressing replication of HCV RNA after being seeded with the exogenous ''anti-HCV'' small interfering RNA (siRNAs) (Kapadia et al 2003;Randall et al 2003;Supekova et al 2008). The exact mechanisms of these complex interactions remain to be better explained.…”