Imai Noriko, Matsuda Noriyuki, Tanaka Keiji, Nakano Akihiko, Matsumoto Shogo, Kang WonKyung
Laboratory of Molecular Entomology and Baculovirology, Wako, Japan.
J Virol. 2003 Jan;77(2):923-30. doi: 10.1128/jvi.77.2.923-930.2003.
The genome of Bombyx mori nucleopolyhedrovirus (BmNPV) is predicted to contain six RING finger proteins: IAP1, ORF35, IAP2, CG30, IE2, and PE38. Several other members of the RING finger family have recently been shown to have the ubiquitin-ligase (E3) activity. We thus examined whether BmNPV RING finger proteins have the E3 activity. In vitro ubiquitination assay with the rabbit reticulocyte lysates and BmNPV RING finger proteins fused with maltose-binding protein (MBP) showed that four of them (IAP2, IE2, PE38, and CG30) were polyubiquitinated in the presence of zinc ion. Furthermore, MBP-IAP2, MBP-IE2, and MBP-PE38 were able to reconstitute ubiquitination activity in cooperation with the Ubc4/5 subfamily of ubiquitin-conjugating enzymes. Mutational analysis also showed that ubiquitination activity of MBP-IAP2, MBP-IE2, and MBP-PE38 were dependent on their RING finger motif. Therefore, these results suggest that IAP2, IE2, and PE38 may function as E3 enzymes during BmNPV infection.
家蚕核型多角体病毒（BmNPV）的基因组预计包含六种环状结构域蛋白：IAP1、ORF35、IAP2、CG30、IE2和PE38。最近有研究表明，环状结构域蛋白家族的其他几个成员具有泛素连接酶（E3）活性。因此，我们检测了BmNPV环状结构域蛋白是否具有E3活性。用兔网织红细胞裂解物和与麦芽糖结合蛋白（MBP）融合的BmNPV环状结构域蛋白进行的体外泛素化试验表明，其中四种蛋白（IAP2、IE2、PE38和CG30）在锌离子存在的情况下被多聚泛素化。此外，MBP-IAP2、MBP-IE2和MBP-PE38能够与泛素结合酶的Ubc4/5亚家族协同重构泛素化活性。突变分析还表明，MBP-IAP2、MBP-IE2和MBP-PE38的泛素化活性依赖于它们的环状结构域基序。因此，这些结果表明，IAP2、IE2和PE38可能在BmNPV感染过程中作为E3酶发挥作用。
