Röttger S, Frischknecht F, Reckmann I, Smith G L, Way M
Cell Biology Programme, European Molecular Biology Laboratory, Heidelberg D-69117, Germany.
J Virol. 1999 Apr;73(4):2863-75. doi: 10.1128/JVI.73.4.2863-2875.1999.
The intracellular enveloped form of vaccinia virus (IEV) induces the formation of actin tails that are strikingly similar to those seen in Listeria and Shigella infections. In contrast to the case for Listeria and Shigella, the vaccinia virus protein(s) responsible for directly initiating actin tail formation remains obscure. However, previous studies with recombinant vaccinia virus strains have suggested that the IEV-specific proteins A33R, A34R, A36R, B5R, and F13L play an undefined role in actin tail formation. In this study we have sought to understand how these proteins, all of which are predicted to have small cytoplasmic domains, are involved in IEV assembly and actin tail formation. Our data reveal that while deletion of A34R, B5R, or F13L resulted in a severe reduction in IEV particle assembly, IEVs formed by the DeltaB5R and DeltaF13L deletion strains, but not DeltaA34R, were still able to induce actin tails. The DeltaA36R deletion strain produced normal amounts of IEV particles, although these were unable to induce actin tails. Using several different approaches, we demonstrated that A36R is a type Ib membrane protein with a large, 195-amino-acid cytoplasmic domain exposed on the surface of IEV particles. Finally, coimmunoprecipitation experiments demonstrated that A36R interacts with A33R and A34R but not with B5R and that B5R forms a complex with A34R but not with A33R or A36R. Using extracts from DeltaA34R- and DeltaA36R-infected cells, we found that the interaction of A36R with A33R and that of A34R with B5R are independent of A34R and A36R, respectively. We conclude from our observations that multiple interactions between IEV membrane proteins exist which have important implications for IEV assembly and actin tail formation. Furthermore, these data suggest that while A34R is involved in IEV assembly and organization, A36R is critical for actin tail formation.
痘苗病毒的细胞内包膜形式（IEV）可诱导肌动蛋白尾的形成，这与在李斯特菌和志贺氏菌感染中观察到的情况极为相似。与李斯特菌和志贺氏菌的情况不同，负责直接启动肌动蛋白尾形成的痘苗病毒蛋白仍不清楚。然而，先前对重组痘苗病毒株的研究表明，IEV特异性蛋白A33R、A34R、A36R、B5R和F13L在肌动蛋白尾形成中发挥着不确定的作用。在本研究中，我们试图了解这些均预测具有小细胞质结构域的蛋白如何参与IEV组装和肌动蛋白尾形成。我们的数据显示，虽然缺失A34R、B5R或F13L会导致IEV颗粒组装严重减少，但由DeltaB5R和DeltaF13L缺失株形成的IEV（而非DeltaA34R）仍能够诱导肌动蛋白尾。DeltaA36R缺失株产生正常数量的IEV颗粒，尽管这些颗粒无法诱导肌动蛋白尾。使用几种不同的方法，我们证明A36R是一种Ib型膜蛋白，具有一个大的、195个氨基酸的细胞质结构域，暴露在IEV颗粒表面。最后，免疫共沉淀实验表明，A36R与A33R和A34R相互作用，但不与B5R相互作用，并且B5R与A34R形成复合物，但不与A33R或A36R形成复合物。使用来自DeltaA34R和DeltaA36R感染细胞的提取物，我们发现A36R与A33R的相互作用以及A34R与B5R的相互作用分别独立于A34R和A36R。我们从观察结果中得出结论，IEV膜蛋白之间存在多种相互作用，这对IEV组装和肌动蛋白尾形成具有重要意义。此外，这些数据表明，虽然A34R参与IEV组装和组织，但A36R对肌动蛋白尾形成至关重要。
