Hartlieb Bettina, Modrof Jens, Mühlberger Elke, Klenk Hans-Dieter, Becker Stephan
Institut für Virologie der Philipps-Universität Marburg, Robert-Koch-Strasse 17, 35037 Marburg, Germany.
J Biol Chem. 2003 Oct 24;278(43):41830-6. doi: 10.1074/jbc.M307036200. Epub 2003 Aug 11.
Transcription of Ebola virus (EBOV)-specific mRNA is driven by the nucleocapsid proteins NP, VP35, and L. This process is further dependent on VP30, an essential EBOV-specific transcription factor. The present study addresses the self-assembly of VP30 and the functional significance of this process for viral transcription and propagation. Essential for oligomerization of VP30 is a region spanning amino acids 94-112. Within this region a cluster of four leucine residues is of critical importance. Mutation of only one of these leucine residues resulted in oligomerization-deficient VP30 molecules that were no longer able to support EBOV-specific transcription. The essential role of homo-oligomerization for the function of VP30 was further corroborated by the finding that mixed VP30 oligomers consisting of VP30 and transcriptionally inactive VP30 mutants were impaired in their ability to support EBOV transcription. The dominant negative effect of these VP30 mutants was dependent on their ability to bind to VP30. The oligomerization of VP30 could be dose dependently inhibited by a 25-mer peptide (E30pep-wt) derived from the presumed oligomerization domain (IC50,1 mum). A control peptide (E30pep-3LA), in which three leucines were changed to alanine, had no inhibitory effect. Thus, E30pep-wt seemed to bind efficiently to VP30 and consequently blocked the oligomerization of the protein. When E30pep-wt was transfected into EBOV-infected cells, the peptide inhibited viral replication suggesting that inhibition of VP30 oligomerization represents a target for EBOV antiviral drugs.
埃博拉病毒（EBOV）特异性mRNA的转录由核衣壳蛋白NP、VP35和L驱动。这一过程进一步依赖于VP30，一种必需的EBOV特异性转录因子。本研究探讨了VP30的自组装及其对病毒转录和传播过程的功能意义。VP30寡聚化所必需的是一个跨越氨基酸94 - 112的区域。在该区域内，四个亮氨酸残基的簇至关重要。仅这些亮氨酸残基中的一个发生突变就会导致寡聚化缺陷的VP30分子，这些分子不再能够支持EBOV特异性转录。由VP30和转录无活性的VP30突变体组成的混合VP30寡聚体在支持EBOV转录的能力上受损，这一发现进一步证实了同型寡聚化对VP30功能的重要作用。这些VP30突变体的显性负效应取决于它们与VP30结合的能力。VP30的寡聚化可以被源自假定寡聚化结构域的25肽（E30pep - wt）剂量依赖性抑制（IC50，1 μM）。一种对照肽（E30pep - 3LA），其中三个亮氨酸被替换为丙氨酸，没有抑制作用。因此，E30pep - wt似乎能有效地与VP30结合，从而阻断该蛋白的寡聚化。当将E30pep - wt转染到EBOV感染的细胞中时，该肽抑制病毒复制，这表明抑制VP30寡聚化是EBOV抗病毒药物的一个靶点。
