乙型肝炎病毒（Hepatitis B virus, HBV）是目前已知的流行范围最广，携带人群最多的病毒之一。世界范围内约20亿人曾感染过HBV，目前约有2.4亿人为慢性HBV携带者，HBV的慢性感染容易发展为严重的肝部疾病如肝硬化甚至肝癌。但针对HBV的感染尚无有效治疗手段。HBV具有极狭窄的宿主范围和嗜肝性，仅能感染肝脏实质细胞，并且天然宿主仅限于人和黑猩猩，这使得HBV的研究很大程度上受限。HBV的入侵特异性与其致病性密切相关，因此HBV入侵特异性的研究对于HBV的致病机理及临床上作为治疗靶点都是非常必要的。钠离子-牛磺胆酸共转运多肽（sodium taurocholate cotransporting polypeptide, NTCP）的发现，为HBV的研究带来了巨大前景。但是HBV的入侵过程目前还并不完全清楚。而HBV作为肝特异性病毒，虽然已知受体NTCP为其嗜肝性的主要决定因素，但其他的入侵特异性机制目前还未得到广泛研究。除受体NTCP外，多种宿主分子也被证实参与HBV的入侵过程。本文从受体NTCP、与受体结合之前病毒的结合分子等多个角度分层面进行了HBV入侵特异性的分子机制研究：

受体作为HBV入侵阶段的最重要分子，不同物种间NTCP的序列差异是导致HBV的宿主限制性的重要原因。啮齿类动物土拨鼠（woodchuck）中天然存在自身嗜肝病毒：土拨鼠肝炎病毒（WHV），过去的研究认为啮齿类动物和灵长类动物之间不能进行嗜肝病毒的跨物种传播，但是随着研究的深入，有报道HBV的卫星病毒HDV感染土拨鼠的例子。我们克隆了土拨鼠的NTCP（wNTCP），发现在人的肝癌细胞HepG2中wNTCP表达和转运功能均与人NTCP（hNTCP）相当。wNTCP可以支持HDV、HBV以及WMHBV的感染。但是感染效率远低于hNTCP。我们进一步对wNTCP进行了研究，发现位于蛋白质第263位的氨基酸序列差异是导致wNTCP不能有效支持感染的主要原因。这部分研究印证了，NTCP作为HBV的功能性受体，是HBV入侵阶段宿主特异性的决定性分子。也提示我们NTCP的保守性有利于正嗜肝病毒属病毒的跨物种传播。

虽然同为肝脏来源的细胞，有些细胞，如原代肝细胞和分化的HepaRG细胞可以支持HBV的感染；有些细胞，如HepG2细胞和未分化的HepaRG细胞则不能支持HBV的感染。实验室早期研究，对比HBV的敏感细胞与HBV的非敏感细胞，找到差异表达的基因，发现除受体NTCP外，还有多种分子参与到HBV的入侵过程，其中之一为凝血因子XII（FXII）。继续研究，我们发现FXII的酶原形式在HBV的入侵过程中起到负调控作用，但是其酶活形式FXIIa则通过桥梁作用帮助HBV入侵原代肝细胞。此外，FXII特定的截短形式也可以促进HBV的感染。FXII在HBV的入侵过程中表现出了双重调控效应，说明血源分子参与并调节HBV的感染，HBV的体内感染情况涉及除受体分子NTCP外更多的调节分子和调控机制。作为血源传播的病毒，HBV在与受体结合之前，首先与宿主体内的血源分子或者组织分子相互作用，这些分子也是决定HBV入侵特异性的重要因素。

Human hepatitis B virus (HBV) is a leading cause of liver diseases ranging from acute hepatitis to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. About 2 billion people have been infected with HBV. With approximately 240 million cases of chronic infection worldwide, HBV is responsible for about 600,000 deaths annually. Current preventive and therapeutic intervention for HBV infection is not adequate. HBV has a narrow host range and a strong tissue tropism. HBV can only infect liver parenchymal cells. And the natural host of HBV is limited within human and chimpanzee. Entry specificity of HBV is the major bottleneck of HBV study and contributes to HBV caused liver pathogenesis. Studies on the molecular mechanisms of HBV entry specificity, will help not only understanding the virus and its pathogenesis, but also developing new therapies for the treatment of the infection and its related diseases. Identification of sodium taurocholate cotransporting polypeptide (NTCP) as the functional receptor of HBV, HDV and WMHBV, has advanced both scientific and clinical understanding for HBV infection. Nevertheless, entry process of HBV remains unclear. Although it is confirmed that NTCP is the major determinant of HBV liver tropism, other mechanisms of HBV entry specificity is not yet well studied. Many other host factors were also found to be involved in HBV entry. In this thesis, we try to explore the molecular determinants of HBV entry specificity.

NTCP orthologs are functionally conserved in mammals. However, variation in their sequences is the major determinant for HBV and HDV infection at entry level among species. Woodchuck (Marmota monax) naturally carries its own hepatitis virus, Woodchuck hepatitis virus (WHV).It was long recognized that species barrier was strictly set between primates and rodents in hepatitis viruses infection, and that no cross-species infection between primates and rodents was occurred naturally. Unexpected, HDV infection of woodchuck in vivo was once reported. To confirm whether woodchuck has the capacity to support HDV infection or not, we successfully cloned woodchuck NTCP (wNTCP) and repeated the infection experiment in vitro. wNTCP expressed well with normal function, and supported HDV, HBV as well as WMHBV infection in human hepatoma cell line, HepG2. HepG2 cells complemented with wNTCP showed much lower efficiency of infection compared to hNTCP. Thoroughly mapping of wNTCP, we found that residue 263 was the major determinant limiting efficiency infection of wNTCP by HDV, HBV and WMHBV. These results confirmed the importance of NTCP for HBV entry specificity. Possibility of hepatitis virus infection in human liver cells was also proved, which is related to the conservation of NTCP orrhologs among species. Thus our results indicated that cross species infection of hepatitis viruses should be taken into concern.

HBV exhibits a very strong tropism for liver parenchymal cells. Primary hepatocytes and differentiated HepaRG cells support HBV infection, while human hepatoma-derived cell line such as HepG2 and undifferentiated HepaRG cells are restricted to HBV infection. HBV-susceptible cell/tissue and HBV unsusceptible cells have distinguished gene expression profiles. Some host genes potentially associated with HBV infection are differentially expressed between HBV-susceptible cells and HBV-unsusceptible cells. From a comparison study of whole genome gene expression array of HBV susceptible and resistant cells, and by siRNA screening, several candidates were selected as potential necessary or inhibitory factors. Among them, coagulation factor XII (FXII) had a strong phenotype. We found FXII was involved in HBV entry as an inhibitory factor in primary tupaia hepatocyte (PTH). We further found that the active form of FXII, FXIIa also influenced HBV entry. The inhibition of FXII was rescued by soluble FXIIa which bound to both PTH and HBV virions. In addition, specific FXII domains also enhanced HBV infection, which showed high affinity to PTH but not HBV virions. Therefore, we propose that FXIIa is likely to function as a bridge between HBV virions and PTH apart from replacement of inhibitor FXII on PTH surface. Our results show that besides receptor, other host factors also modulate and control the specificity of HBV entry.