Role of HIV-1 Nef in Acceleration of HCV-Mediated Liver Disease

描述(由申请人提供)：HIV-1已知通过通过尚不清楚的机制加速丙型肝炎病毒复制而加重丙型肝炎病毒相关的肝病。最近的研究表明，HIV-1Nef可以从HIV-1敏感细胞转移到其他未感染的敏感细胞，甚至通过管道或外切体转移到非敏感细胞。此外，Nef还具有多种加重功能，包括细胞内脂质介导的丙型肝炎病毒复制，与调控癌症的细胞激酶的复合体，以及干扰抗丙型肝炎病毒宿主的免疫防御。因此，我们研究了Nef在丙型肝炎病毒感染的肝细胞中的作用，以更好地了解合并感染的病理生物学。我们的数据显示，肝细胞不支持HIV复制。没有观察到病毒复制的证据，即使当HIV-1转基因的肝细胞与Jurkat T细胞共同培养时，也表明混合感染患者的肝脏恶化不是由于HIV-1在感染丙型肝炎病毒的宿主的肝细胞中复制所致。相反，HIV-1Nef蛋白通过管道从表达NEF的T细胞转移到肝细胞，通过与表达NEF的Jurkat细胞共同培养24小时，高达16%的肝细胞携带转移的Nef。此外，Nef改变了脂滴(LD)的大小和数量，并持续上调目标肝细胞中丙型肝炎病毒复制1.5~2.5倍，考虑到另一种缓慢的基线复制，这一点意义重大。NEF还显著增加了ROS的产生，ROS可以激活信号分子，如MAP激酶，诱导转化生长因子�1的表达。此外，Nef还增加了清道夫受体B1(SR-B1)在细胞内和细胞表面的表达，而SR-B1是Huh7.5.1中病毒进入和胆固醇转运所必需的，这表明Nef可以促进非丙型肝炎病毒感染的肝细胞对丙型肝炎病毒感染的易感性。此外，Nef增强了乙醇介导的丙型肝炎病毒复制上调，从而加速了肝细胞癌的发生。综上所述，这些数据表明，HIV-1Nef是通过促进丙型肝炎病毒复制和协调调节细胞内和细胞外的关键分子而加速丙型肝炎病毒介导的肝脏发病的关键因素。基于这些初步发现，我们为这个项目提出了三个具体目标。成功地实现这些目标将阐明HIV-1介导的肝脏恶化的病理生物学机制，从而为双重病毒肝病的预后和治疗提供更有效的工具。

DESCRIPTION (provided by applicant): HIV-1 is known to aggravate HCV-related liver disease by accelerating HCV replication through as yet unclear mechanisms. Recent studies indicate that HIV-1 Nef can be transferred from HIV-1 susceptible cells to other uninfected susceptible and even to non-susceptible cells through conduits or exosomes. Further, Nef possesses multiple exacerbating functions including intracellular lipid mediation of HCV replication, complexing with cancer-modulating cellular kinases, and interference with anti-HCV host immune defenses. Accordingly, we have studied the role of Nef in HCV-infected hepatocytes to better understand the pathobiology of co- infection. Our data showed that hepatocytes do not support HIV replication. No evidence of virus replication was observed, even when the HIV-1-transfected hepatocytes were co-cultured with Jurkat T cells, indicating that liver deterioration in the co-infected patient is not due to the replication of HIV-1 in the hepatocytes of the HCV infected host. Instead, HIV-1 Nef protein was found to be transferred from expressing T cells to hepatocytes through conduits, wherein up to 16% of hepatocytes harbor the transferred Nef by co-cultivation with nef-expressing Jurkat cells for 24 hr. Moreover, Nef altered the size and numbers of lipid droplets (LD) and consistently up-regulated HCV replication by 1.5~2.5 fold in the target hepatocytes, which is significant in view of the otherwis indolent baseline replication. Nef also dramatically augmented reactive oxygen species (ROS) production, which can activate signaling molecules, such as MAP kinase, to induce TGF�1 expression. Besides, Nef increased intracellular and cell surface expression of scavenger receptor B1 (SR-B1), which is integral for virus entry and cholesterol trafficking in Huh7.5.1, implying that Nef can foster susceptibility of HCV infection in non-HCV infected hepatocytes. Further, Nef enhanced ethanol-mediated up-regulation of HCV replication so as to accelerate hepatocellular carcinoma (HCC). Taken together, these data indicate that HIV-1 Nef is a critical element in accelerating HCV-mediated liver pathogenesis via enhancing HCV replication and coordinating modulation of key intra- and extra-cellular molecules for liver decay. Based on these preliminary findings, we propose three Specific Aims for this project. Success in achieving these goals will clarify the pathobiologic mechanisms of HIV-1-mediated exacerbation of liver decay, leading to more effective tools for prognosis and therapeutics against dual virus hepatic disease.