Moraleda G, Seeholzer S, Bichko V, Dunbrack R, Otto J, Taylor J
Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111-2497, USA.
J Virol. 1999 Sep;73(9):7147-52. doi: 10.1128/JVI.73.9.7147-7152.1999.
The large form of the hepatitis delta virus (HDV) protein (L) can be isoprenylated near its C terminus, and this modification is considered essential for particle assembly. Using gel electrophoresis, we separated L into two species of similar mobilities. The slower species could be labeled by the incorporation of [(14)C]mevalonolactone and is interpreted to be isoprenylated L (L(i)). In serum particles, infected liver, transfected cells, and assembled particles, 25 to 85% of L was isoprenylated. Isoprenylation was also demonstrated by (14)C incorporation in vitro with a rabbit reticulocyte coupled transcription-translation system. However, the species obtained migrated even slower than that detected by labeling in vivo. Next, in studies of HDV particle assembly in the presence of the surface proteins of human hepatitis B virus, we observed the following. (i) Relative to L, L(i) was preferentially assembled into virus-like particles. (ii) L(i) could coassemble the unmodified L and the small delta protein, S. (iii) In contrast, a form of L with a deletion in the dimerization domain was both isoprenylated and assembled, but it could not support the coassembly of S. Finally, to test the expectation that the isoprenylation of L would increase its hydrophobicity, we applied a phase separation strategy based on micelle formation with the nonionic detergent Triton X-114. We showed the following. (i) The unique C-terminal 19 amino acids present on L relative to S caused a significant increase in the hydrophobicity. (ii) This increase was independent of isoprenylation. (iii) In contrast, other, artificial modifications at either the N or C terminus of S did not increase the hydrophobicity. (iv) The increased hydrophobicity was not sufficient for particle assembly; nevertheless, we speculate that it might facilitate virion assembly.
丁型肝炎病毒（HDV）的大蛋白形式（L）在其C末端附近可被异戊二烯化，这种修饰被认为对病毒颗粒组装至关重要。我们利用凝胶电泳将L分离为两种迁移率相似的蛋白。迁移较慢的蛋白可通过掺入[(14)C]甲羟戊酸内酯进行标记，并被解释为异戊二烯化的L（L(i)）。在血清颗粒、受感染的肝脏、转染细胞和组装颗粒中，25%至85%的L被异戊二烯化。在体外利用兔网织红细胞偶联转录-翻译系统进行的(14)C掺入实验也证实了异戊二烯化的存在。然而，体外获得的蛋白迁移速度比体内标记检测到的还要慢。接下来，在研究人乙型肝炎病毒表面蛋白存在下的HDV颗粒组装时，我们观察到以下情况。（i）相对于L，L(i)优先组装成病毒样颗粒。（ii）L(i)可以与未修饰的L和小δ蛋白S共同组装。（iii）相比之下，二聚化结构域缺失的L形式既被异戊二烯化又能组装，但它不能支持S的共同组装。最后，为了验证L的异戊二烯化会增加其疏水性这一预期，我们应用了基于非离子去污剂Triton X-114形成胶束的相分离策略。我们发现以下情况。（i）相对于S，L上独特的C末端19个氨基酸导致疏水性显著增加。（ii）这种增加与异戊二烯化无关。（iii）相比之下，S的N末端或C末端的其他人工修饰并未增加疏水性。（iv）增加的疏水性不足以进行颗粒组装；不过，我们推测它可能有助于病毒粒子的组装。
