Alaoui-Ismaili M H, Richardson C D
Amgen Research Institute, Toronto, Ontario, Canada.
J Virol. 1998 Mar;72(3):2213-23. doi: 10.1128/JVI.72.3.2213-2223.1998.
Entomopoxviruses and baculoviruses are pathogens of insects which replicate in the cytoplasm and nuclei of their host cells, respectively. During the late stages of infection, both groups of viruses produce occlusion bodies which serve to protect virions from the external environment. Immunofluorescence and electron microscopy studies have shown that large bundles of filaments are associated with these occlusion bodies. Entomopoxviruses produce cytoplasmic fibrils which appear to be composed of the filament-associated late protein of entomopoxviruses (FALPE). Baculoviruses, on the other hand, yield filaments in the nuclei and cytoplasm of the infected cell which are composed of a protein called p10. Despite significant differences in their sequences, FALPE and p10 have similar hydrophilicity profiles, and each has a proline-rich stretch of amino acids at its carboxyl terminus. Evidence that FALPE and p10 could produce filaments in the absence of other viral proteins is presented. When FALPE was expressed in insect cells from a recombinant baculovirus, filaments similar to those produced by the wild-type Amsacta moorei entomopoxvirus were observed. In addition, when expression plasmids containing FALPE or p10 genes were transfected into Vero monkey kidney cells, filament structures similar to those found in infected insect cells were produced. The manner in which FALPE and p10 subunits interact to form polymers was investigated through deletion and site-specific mutagenesis in conjunction with immunofluorescence microscopy, yeast two-hybrid protein interaction analysis, and chemical cross-linking of adjacent molecules. These studies indicated that the amino termini of FALPE and p10 were essential for subunit interaction. Although deletion of the carboxy termini did not affect this interaction, it did inhibit filament formation. In addition, modification of several potential sites for phosphorylation also abolished filament assembly. We concluded that although the sequences of FALPE and p10 were different, the structural and functional properties of the two polypeptides appeared to be similar.
昆虫痘病毒和杆状病毒是昆虫病原体，分别在宿主细胞的细胞质和细胞核中复制。在感染后期，这两类病毒都会产生包涵体，用于保护病毒粒子免受外部环境影响。免疫荧光和电子显微镜研究表明，大量的丝状束与这些包涵体相关。昆虫痘病毒产生细胞质纤维，似乎由昆虫痘病毒丝状相关晚期蛋白（FALPE）组成。另一方面，杆状病毒在受感染细胞的细胞核和细胞质中产生由一种名为p10的蛋白质组成的细丝。尽管FALPE和p10的序列存在显著差异，但它们具有相似的亲水性图谱，并且在其羧基末端都有一段富含脯氨酸的氨基酸序列。本文提供了FALPE和p10在没有其他病毒蛋白的情况下能够产生细丝的证据。当FALPE通过重组杆状病毒在昆虫细胞中表达时，观察到了与野生型摩尔纹灯蛾昆虫痘病毒产生的细丝相似的细丝。此外，当将含有FALPE或p10基因的表达质粒转染到非洲绿猴肾细胞中时，产生了与在受感染昆虫细胞中发现的细丝结构相似的细丝。通过缺失和位点特异性诱变，结合免疫荧光显微镜、酵母双杂交蛋白相互作用分析以及相邻分子的化学交联，研究了FALPE和p10亚基相互作用形成聚合物的方式。这些研究表明，FALPE和p10的氨基末端对于亚基相互作用至关重要。虽然羧基末端的缺失不影响这种相互作用，但它确实抑制了细丝的形成。此外，对几个潜在磷酸化位点的修饰也消除了细丝组装。我们得出结论，尽管FALPE和p10的序列不同，但这两种多肽的结构和功能特性似乎相似。
