ANALYSIS OF VIRAL REPLICATION AND INFECTION MECHANISM OF THE DUCK HEPATITIS B VIRUS

为了确定DHBV的候选受体，我们先前已经确定了一种180 kDa的糖蛋白，它与DHBV颗粒和大肠杆菌来源的GST-PreS多肽结合。Gp18O与已知序列的序列比较表明，它代表了一个新的膜结合羧肽酶家族的原型。初步的序列分析表明，CPD由三个腔/胞外羧肽酶B或H结构域(称为A、B和C)、一个疏水性跨膜锚定和一个高度保守的细胞质尾部组成。结构域C已被阐明具有DHBVPreS结合部位。目前，gp180是一种驻留在高尔基体上的蛋白，被认为是禽流感病毒的细胞受体，介导病毒附着和内化到宿主细胞中。所有的乙肝病毒(HBV)在感染过程中都表现出明显的物种特异性。我们用鸭乙肝病毒(D乙肝)和苍鹭乙肝病毒(H乙肝)作为模型系统，研究了这种狭窄宿主范围的分子基础。我们先前已经发现，在禽流感病毒中，大病毒包膜蛋白的PreS结构域决定了宿主范围，这是通过将HHBVenv与DHBV/HHBV嵌合包膜蛋白进行伪分型来实现的。为了进一步定位DHBVPreS区的决定簇，我们制作了DHBV/HHBV嵌合包膜蛋白(HDC 6-12)。仅需15个氨基酸的替换，就足以感染鸭的肝细胞。该结构域位于DHBVPreS序列中的22-37位氨基酸之间，位于鸭羧肽酶D(DCPD)结合位点(43-108氨基酸)的更多氨基末端区域之外，提示DCPD不是病毒进入的唯一决定因素，该结构域可能与可能的第二受体结合有关。

In an attempt to identify receptor candidates for DHBV, we have previously identified a glycoprotein of 180 kDa, which binds DHBV particles and Escherichia coli-derived GST-preS polypeptides. Sequence comparisons of gp18O cDNA with known sequences suggested that it represents the prototype of a new family of membrane bound carboxypeptidases. The primary sequence analysis indicates that CPDs consist of three luminal/extracellular carboxypeptidase B or H like domains (called A, B, and C), a hydrophobic transmembrane anchor and a highly conserved cytoplasmic tail. Domain C has been elucidated to have the DHBV preS-binding site. Now gp180, a Golgi-resident protein, is thought to be the cellular receptor for avian hepadnaviruses that mediates virus attachment and internalization into the host cell.All hepatitis B viruses (HBVs) display marked species specificity in their infection. We have examined the molecular basis for this narrow host range, using duck HBV (DHBV) and heron HBV (HHBV) as a model system. We have previously revealed that the preS domain of the large viral envelope protein determines host range in avian hepatitis B viruses, by pseudotyping of HHBV env with DHBV/HHBV chimeric envelope proteins. To further map the determinant in the DHBV preS region, we have made DHBV/HHBV chimeric envelope proteins (HDC 6-12). The replacement of as few as 15 amino acids of the preS domain of the HHBV L protein by their counterparts is sufficient to permit infection of duck hepatocytes. This domain is located between amino acid 22 and 37 in the DHBV preS sequence, which is outside of and more amino terminal domain of duck carboxypeptidase D (dCPD) binding sites (amino acid 43-108).These results suggest that dCPD could not be the sole determinant of viral entry, and this domain might be responsible for the binding of the putative second receptor in hepadnaviral infection.