Sehgal Deepak, Thomas Saijo, Chakraborty Mahua, Jameel Shahid
Virology Group, International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
Virol J. 2006 May 26;3:38. doi: 10.1186/1743-422X-3-38.
The ORF1 of hepatitis E virus (HEV) encodes a nonstructural polyprotein of approximately 186 kDa that has putative domains for four enzymes: a methyltransferase, a papain-like cysteine protease, a RNA helicase and a RNA dependent RNA polymerase. In the absence of a culture system for HEV, the ORF1 expressed using bacterial and mammalian expression systems has shown an approximately 186 kDa protein, but no processing of the polyprotein has been observed. Based on these observations, it was proposed that the ORF1 polyprotein does not undergo processing into functional units. We have studied ORF1 polyprotein expression and processing through a baculovirus expression vector system because of the high level expression and post-translational modification abilities of this system.
The baculovirus expressed ORF1 polyprotein was processed into smaller fragments that could be detected using antibodies directed against tags engineered at both ends. Processing of this approximately 192 kDa tagged ORF1 polyprotein and accumulation of lower molecular weight species took place in a time-dependent manner. This processing was inhibited by E-64d, a cell-permeable cysteine protease inhibitor. MALDI-TOF analysis of a 35 kDa processed fragment revealed 9 peptide sequences that matched the HEV methyltransferase (MeT), the first putative domain of the ORF1 polyprotein. Antibodies to the MeT region also revealed an ORF1 processing pattern identical to that observed for the N-terminal tag.
When expressed through baculovirus, the ORF1 polyprotein of HEV was processed into smaller proteins that correlated with their proposed functional domains. Though the involvement of non-cysteine protease(s) could not be be ruled out, this processing mainly depended upon a cysteine protease.
戊型肝炎病毒（HEV）的开放阅读框1（ORF1）编码一种约186 kDa的非结构多聚蛋白，该蛋白具有四种酶的假定结构域：一种甲基转移酶、一种木瓜蛋白酶样半胱氨酸蛋白酶、一种RNA解旋酶和一种RNA依赖性RNA聚合酶。在缺乏HEV培养系统的情况下，使用细菌和哺乳动物表达系统表达的ORF1显示出一种约186 kDa的蛋白，但未观察到多聚蛋白的加工过程。基于这些观察结果，有人提出ORF1多聚蛋白不会加工成功能单元。由于杆状病毒表达载体系统具有高水平表达和翻译后修饰能力，我们通过该系统研究了ORF1多聚蛋白的表达和加工过程。
杆状病毒表达的ORF1多聚蛋白被加工成较小的片段，这些片段可以使用针对两端工程化标签的抗体进行检测。这种约192 kDa的带标签ORF1多聚蛋白的加工过程以及低分子量物种的积累呈时间依赖性。这种加工过程被E-64d（一种细胞可渗透的半胱氨酸蛋白酶抑制剂）抑制。对一个35 kDa加工片段的基质辅助激光解吸电离飞行时间（MALDI-TOF）分析揭示了9个肽序列，这些序列与HEV甲基转移酶（MeT）匹配，MeT是ORF1多聚蛋白的第一个假定结构域。针对MeT区域的抗体也显示出与N端标签观察到的相同的ORF1加工模式。
当通过杆状病毒表达时，HEV的ORF1多聚蛋白被加工成与其假定功能结构域相关的较小蛋白质。虽然不能排除非半胱氨酸蛋白酶的参与，但这种加工主要依赖于一种半胱氨酸蛋白酶。
