Species-Specific Regions of Occludin Required by Hepatitis C Virus for Cell Entry
Species-Specific Regions of Occludin Required by Hepatitis C Virus for Cell Entry
复制标题
DOI：
10.1128/jvi.01555-10
复制
发表时间：
2010-11-01
影响因子：
5.4
通讯作者：
Evans, Matthew J.
中科院分区：
文献类型：
--
作者：
Michta, Maria L.;Hopcraft, Sharon E.;Evans, Matthew J.
丙型肝炎病毒（HCV）是全球肝脏疾病的主要原因。由于丙型肝炎病毒只感染人类和黑猩猩细胞，由于缺乏方便的小动物模型，抗病毒治疗和疫苗的开发受到了阻碍。在这项研究中，我们进一步研究了HCV的物种趋向性是如何在细胞进入水平上被调节的。先前已经确定紧密连接蛋白（OCLN）对HCV宿主细胞进入至关重要，并且人类OCLN在介导HCV细胞进入方面比小鼠同源物更有效。为了进一步研究OCLN序列与HCV物种趋向性之间的关系，我们比较了来自一系列物种的OCLN蛋白介导携带HCV糖蛋白的慢病毒假颗粒感染天然OCLN缺陷786-O细胞的能力。虽然灵长类动物的OCLN序列与人类的功能相当，但犬、仓鼠和大鼠的OCLN具有中间活性，而豚鼠的OCLN完全没有功能。通过分析这些OCLN蛋白之间的嵌合体和OCLN细胞外结构域的丙氨酸扫描诱变，我们确定了第二个细胞外环（EC2）的后半部分和该结构域内的特定氨基酸对调节该蛋白的HCV细胞进入因子活性至关重要。此外，EC2的这个关键区域两侧有两个保守的半胱氨酸残基，这是HCV进入细胞所必需的，这表明EC2的一个亚结构域可能是由二硫键定义的。
Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. As HCV infects only human and chimpanzee cells, antiviral therapy and vaccine development have been hampered by the lack of a convenient small-animal model. In this study we further investigate how the species tropism of HCV is modulated at the level of cell entry. It has been previously determined that the tight junction protein occludin (OCLN) is essential for HCV host cell entry and that human OCLN is more efficient than the mouse ortholog at mediating HCV cell entry. To further investigate the relationship between OCLN sequence and HCV species tropism, we compared OCLN proteins from a range of species for their ability to mediate infection of naturally OCLN-deficient 786-O cells with lentiviral pseudoparticles bearing the HCV glycoproteins. While primate sequences function equivalently to human OCLN, canine, hamster, and rat OCLN had intermediate activities, and guinea pig OCLN was completely nonfunctional. Through analysis of chimeras between these OCLN proteins and alanine scanning mutagenesis of the extracellular domains of OCLN, we identified the second half of the second extracellular loop (EC2) and specific amino acids within this domain to be critical for modulating the HCV cell entry factor activity of this protein. Furthermore, this critical region of EC2 is flanked by two conserved cysteine residues that are essential for HCV cell entry, suggesting that a subdomain of EC2 may be defined by a disulfide bond.