Husain Matloob, Moss Bernard
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Virol. 2002 Aug;76(15):7777-89. doi: 10.1128/jvi.76.15.7777-7789.2002.
Intracellular mature vaccinia virions are wrapped by cisternae, derived from virus-modified trans-Golgi or endosomal membranes, and then transported via microtubules to the cell periphery. Two viral proteins, encoded by the F13L and B5R open reading frames, are essential for the membrane-wrapping step. Previous transfection studies indicated that F13L induces the formation of post-Golgi vesicles that incorporate the B5R protein and that this activity depends on an intact F13L phospholipase motif. Here we show that the F13L protein has a general effect on the trafficking of integral membrane proteins from the Golgi apparatus, as both the vaccinia virus A36R protein and the vesicular stomatitis virus G protein also colocalized with the F13L protein in vesicles. In addition, increased expression of cellular phospholipase D, which has a similar phospholipase motif as, but little amino acid sequence identity with, F13L, induced post-Golgi vesicles that contained B5R and A36R proteins. Butanol-1, which prevents the formation of phosphatidic acid by phospholipase D and specifically inhibits phospholipase D-mediated vesicle formation, also inhibited F13L-induced vesicle formation, whereas secondary and tertiary alcohols had no effect. Moreover, inhibition of phospholipase activity by butanol-1 also reduced plaque size and decreased the formation of extracellular vaccinia virus without affecting the yield of intracellular mature virus. Phospholipase D, however, could not complement a vaccinia virus F13L deletion mutant, indicating that F13L has additional virus-specific properties. Taken together, these data support an important role for F13L in inducing the formation of vesicle precursors of the vaccinia virus membrane via phospholipase activity or activation.
细胞内成熟的痘苗病毒粒子被源自病毒修饰的反式高尔基体或内体膜的扁平囊所包裹，然后通过微管运输到细胞周边。由F13L和B5R开放阅读框编码的两种病毒蛋白对于膜包裹步骤至关重要。先前的转染研究表明，F13L诱导包含B5R蛋白的高尔基体后囊泡的形成，并且这种活性依赖于完整的F13L磷脂酶基序。在这里我们表明，F13L蛋白对来自高尔基体的整合膜蛋白的运输具有普遍影响，因为痘苗病毒A36R蛋白和水疱性口炎病毒G蛋白也与F13L蛋白在囊泡中共定位。此外，细胞磷脂酶D的表达增加，其具有与F13L相似的磷脂酶基序，但氨基酸序列同一性很少，诱导了包含B5R和A36R蛋白的高尔基体后囊泡。1-丁醇可阻止磷脂酶D形成磷脂酸并特异性抑制磷脂酶D介导的囊泡形成，也抑制F13L诱导的囊泡形成而仲醇和叔醇则没有作用。此外，1 - 丁醇对磷脂酶活性的抑制也减小了蚀斑大小并减少了细胞外痘苗病毒的形成，而不影响细胞内成熟病毒产量。然而磷脂酶D不能补充痘苗病毒F13L缺失突变体，表明F13L具有额外的病毒特异性特性。综上所述，这些数据支持F13L在通过磷脂酶活性或激活诱导痘苗病毒膜的囊泡前体形成中起重要作用。
