Hepatitis C Virus and Hepatocellular Carcinoma

描述(申请人提供)：干扰素(干扰素-α)和利巴韦林是慢性丙型肝炎病毒感染患者的标准治疗方法，但这种治疗方法无法清除大多数患者的病毒。对干扰素-α/利巴韦林无应答的患者发展为肝硬变和癌症的风险很高。丙型肝炎病毒感染细胞对外源性干扰素-α/利巴韦林产生耐药性的机制尚不清楚。这笔赠款的长期目标是了解干扰素-α/利巴韦林联合治疗的耐药机制，以便开发有效的策略来清除慢性感染患者的病毒，防止肝硬化和肝细胞癌的发展。我们产生了以下结果，支持新的具体目标。(I)。在这项应用的最后一次审查中，我们开发了一种稳定的、持续感染丙型肝炎病毒的Huh-7细胞培养系统，具有非常高的病毒滴度(&gt；100天)。采用长期治疗方法，我们发现干扰素-λ有效地清除了持续感染细胞中的丙型肝炎病毒复制，而持续感染的培养细胞中的丙型肝炎病毒复制仍然对干扰素-α和利巴韦林联合治疗耐药。(Ii)。我们发现，丙型肝炎病毒感染可诱导内质网应激和自噬反应，下调干扰素α受体1(IFNAR1)的表达，导致Jak-Stat信号通路缺陷，STAT1、STAT2的磷酸化/核转位减少，抗病毒反应减弱。(三)。我们的结果表明，利巴韦林和干扰素-α在感染的非常早期进行治疗时，可以协同抑制病毒复制。 病毒滴度低的培养。每种药物都通过两种不同的机制阻止多聚核糖体加载到丙型肝炎病毒IRES mRNA上，从而抑制翻译。此外，我们发现持续感染的丙型肝炎病毒细胞由于对利巴韦林的摄取受损而对利巴韦林产生抗药性。(四)。我们发现丙型肝炎病毒感染不会改变干扰素-λ受体-1的表达。干扰素-λ激活STAT3、AKT和MAPK途径抑制丙型肝炎病毒复制，克服干扰素-α/利巴韦林耐药机制，提示III型干扰素系统在清除丙型肝炎病毒感染中起着非常重要的作用。我们的结果表明，干扰素-λ抗丙型肝炎病毒的机制是新的，并且超越了经典的JAK-STAT信号。这项拨款申请中提出的研究将使用应答者和无应答者的慢性感染的人肝细胞来验证干扰素-α/利巴韦林耐药机制。我们还将探索III型干扰素(IL-29、IL-28A和IL-28B)抑制干扰素-a耐药细胞中丙型肝炎病毒复制的独特抗病毒机制。我们的新假设是，丙型肝炎病毒感染肝细胞后触发内质网应激反应，从而下调干扰素α受体1的表达，导致JAK-STAT信号通路缺陷、慢性感染和对干扰素-α的抗病毒反应减弱。我们推测，由于干扰素-λ强烈抑制丙型肝炎病毒在持续感染细胞中的复制，探索其生物学作用和抗病毒机制将为无应答的慢性丙型肝炎患者提供一种替代治疗方法。这一假设将在以下三个具体目标下进行检验。在特定的目标1中，我们将确定在丙型肝炎病毒感染的人肝脏中Jak-Stat信号缺陷的机制。在具体目标2中，我们将研究利巴韦林的协同作用和耐药机制。 丙型肝炎病毒感染。在具体目标3中，我们将建立不同的信号转导和抗病毒基因调控机制，通过这些机制，干扰素-λ可以有效地清除JAK-STAT信号缺陷的Huh-7细胞中的丙型肝炎病毒。如果这些实验成功，将增加我们对干扰素耐药机制的了解，克服耐药性，清除感染，预防肝细胞癌。

DESCRIPTION (provided by applicant): Interferon alpha (IFN-α) along with ribavirin is the standard treatment for patients with chronic HCV infection but this therapy is unable to clear the virus in the majority of patients. Patients who remain non-responders to IFN-α /ribavirin are at a high risk of developing liver cirrhosis and cancer. The mechanisms by which HCV infected cells develop resistance to exogenous IFN-α/ribavirin are unknown. The long-term goal of this grant is to understand the resistance mechanisms of IFN-α/ribavirin combination treatment so that effective strategies can be developed to clear the virus in chronically infected patients and prevent development of liver cirrhosis and hepatocellular carcinoma. We generated the following results that support the new Specific Aims. (i). During the last review of this applicatin we have developed a stable and an HCV persistently infected Huh-7 cell culture system with very high viral titer (>100 days). Using a long-term treatment approach we showed that interferon lambda (IFN-λ) effectively clears the HCV replication from persistently infected cells whereas HCV replication in the persistently infected culture cells remained resistant to IFN-α and ribavirin combination treatment. (ii). We published that HCV infection induces ER-stress and autophagy response and down regulates the expression of interferon alpha-receptor 1 (IFNAR1) leading to a defective Jak-Stat signaling, reduced phosphorylation /nuclear translocation of Stat1, Stat2 and impaired antiviral response. (iii). Our results show that ribavirn and IFN-α synergistically inhibit virus replication when treated at very early stage of an infected culture with low viral titer. Each agent inhibits translation by preventing the polyribosomes load to the HCV IRES mRNA using two separate mechanisms. Furthermore, we found that persistently infected HCV cells develop resistance to ribavirin due to impaired uptake of ribavirin. (iv). We showed that HCV infection does not alter IFN-λ receptor-1 expression. IFN-λ activates Stat3, AKT and MAPK pathways to inhibit HCV replication and overcomes IFN-α /ribavirin resistance mechanisms suggesting that type III interferon system plays a very important role in the clearance of hepatitis C virus infection. Our results suggest that the IFN-λ antiviral mechanism against HCV is novel and operates beyond the classical Jak-Stat signaling. Studied proposed in this grant application will validate IFN-α/ribavirin resistant mechanisms using chronically infected human hepatocytes from responders and non-responders. We will also explore the unique antiviral mechanism by which type III interferon (IL-29, IL-28A and IL-28B) inhibits HCV replication in IFN-a resistant cells. Our new hypothesis is that HCV infection of hepatocytes triggers the ER stress response and thereby down regulates the expression of interferon alpha receptor 1 (IFNAR1) leading to the defective Jak-Stat signaling, chronic infection and an impaired antiviral response to IFN-α. We postulate that since IFN-λ strongly inhibits HCV replication in persistently infected cells, exploring its biological role and antivira mechanisms should provide an alternative therapy for chronic HCV patients who are non-responders. The hypothesis will be tested under the following three Specific Aims. In Specific Aim 1, we will determine the mechanism of defective Jak-Stat signaling in HCV infected human liver. In Specific Aim 2, we will investigate the synergy and resistance mechanisms of ribavirin in HCV infection. In Specific Aim 3, we will establish the distinct signal transduction and antiviral gene regulation mechanisms by which IFN-λ effectively clears HCV in Huh-7 cells with defective Jak-Stat signaling. If these experiments are successful it will increase our understanding of the IFN-resistance mechanisms, overcome the resistance and clear the infection and prevent hepatocellular carcinoma.