Le Seyec J, Chouteau P, Cannie I, Guguen-Guillouzo C, Gripon P
Unité de Recherches Hépatologiques U 49, Institut National de la Santé et de la Recherche Médicale, Hôpital de Pontchaillou, 35033 Rennes Cedex, France.
J Virol. 1999 Mar;73(3):2052-7. doi: 10.1128/JVI.73.3.2052-2057.1999.
During the life cycle of hepatitis B virus (HBV), the large envelope protein (L) plays a pivotal role. Indeed, this polypeptide is essential for viral assembly and probably for the infection process. By performing mutagenesis experiments, we have previously excluded a putative involvement of the pre-S2 domain of the L protein in viral infectivity. In the present study, we have evaluated the role of the pre-S1 region in HBV infection. For this purpose, 21 mutants of the L protein were created. The entire pre-S1 domain was covered by contiguous deletions of 5 amino acids. First, after transfection into HepG2 cells, the efficient expression of both glycosylated and unglycosylated L mutant proteins was verified. The secretion rate of envelope proteins was modified positively or negatively by deletions, indicating that the pre-S1 domain contains several regulating sequences able to influence the surface protein secretion. The ability of mutant proteins to support the production of virions was then studied. Only the four C-terminal deletions, covering the 17 amino acids suspected to interact with the cytoplasmic nucleocapsids, inhibited virion release. Finally, the presence of the modified pre-S1 domain at the external side of all secreted virions was confirmed, and their infectivity was assayed on normal human hepatocytes in primary culture. Only a short sequence including amino acids 78 to 87 tolerates internal deletions without affecting viral infectivity. These results confirm the involvement of the L protein in the infection step and demonstrate that the sequence between amino acids 3 and 77 is involved in this process.
在乙型肝炎病毒（HBV）的生命周期中，大包膜蛋白（L）起着关键作用。实际上，这种多肽对于病毒组装以及可能对于感染过程都是必不可少的。通过进行诱变实验，我们先前已排除L蛋白的前S2结构域在病毒感染性中的假定作用。在本研究中，我们评估了前S1区域在HBV感染中的作用。为此，创建了21个L蛋白突变体。整个前S1结构域被5个氨基酸的连续缺失所覆盖。首先，转染到HepG2细胞后，验证了糖基化和非糖基化L突变蛋白的有效表达。缺失对包膜蛋白的分泌率有正向或负向的影响，表明前S1结构域包含几个能够影响表面蛋白分泌的调控序列。然后研究了突变蛋白支持病毒粒子产生的能力。只有覆盖怀疑与细胞质核衣壳相互作用的17个氨基酸的四个C末端缺失抑制了病毒粒子的释放。最后，证实了所有分泌的病毒粒子外侧存在修饰的前S1结构域，并在原代培养的正常人肝细胞上测定了它们的感染性。只有包含氨基酸78至87的短序列能够耐受内部缺失而不影响病毒感染性。这些结果证实了L蛋白参与感染步骤，并证明氨基酸3至77之间的序列参与了这一过程。
