Nakayama Tetsuo, Komase Katsuhiro, Uzuka Rina, Hoshi Akiyoshi, Okafuji Takao
Department of Virology, Center for Basic Research1, and Division of Research and Development, Research Center for Biologicals2, The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan.
Department of Pediatrics, Tokyo Medical University, 6-7-1 Nishishinjyuku, Shinjyuku-ku, Tokyo 160-0023, Japan3.
J Gen Virol. 2001 Sep;82(Pt 9):2143-2150. doi: 10.1099/0022-1317-82-9-2143.
The live measles virus (MV) vaccine strain AIK-C was attenuated from the wild-type strain Edmonston by plaque purification at 33 degrees C. Strain AIK-C grew well at 33 degrees C with a mixture of small-and medium-sized plaques in Vero cells, but did not grow well at 40 degrees C. To investigate fusion inducibility, expression plasmids for the fusion (F) and haemagglutinin (H) protein regions of MV strains AIK-C (pAIK-F01 and pAIK-H) and Edmonston (pEdm-F and pEdm-H) were constructed. pEdm-F induced extensive cell fusion in B95a and Vero cells under the control of T7 RNA polymerase, whereas a sharp reduction in syncytium formation was observed when pAIK-F01 was used. Six amino acid differences were determined between pAIK-F01 and pEdm-F. Direct sequencing showed that the seed strain AIK-C contained either Leu or Phe at position 278 of the F protein. Experiments using recombinant F protein plasmids demonstrated that those with Leu at position 278 induced poor syncytium formation, while those with Phe at position 278 (Edmonston type) induced extensive cell fusion. Replacement of Phe with Leu at position 278 of pEdm-F reduced fusion-inducing capability. A full-length infectious clone of AIK-C with Leu at position 278 of the F protein was constructed. The rescued virus produced small plaques in Vero cells. However, the same rescued virus with Phe at position 278 produced large plaques. It was concluded that Leu at position 278 of the F protein of the MV vaccine strain AIK-C is responsible for the formation of small plaques.
减毒活麻疹病毒（MV）疫苗株AIK - C是通过在33℃下空斑纯化从野生型Edmonston株中减毒而来的。AIK - C株在33℃下于Vero细胞中生长良好，形成大小不一的空斑，但在40℃下生长不佳。为了研究融合诱导性，构建了MV株AIK - C（pAIK - F01和pAIK - H）及Edmonston（pEdm - F和pEdm - H）的融合（F）蛋白和血凝素（H）蛋白区域的表达质粒。在T7 RNA聚合酶控制下，pEdm - F在B95a和Vero细胞中诱导广泛的细胞融合，而使用pAIK - F01时则观察到多核体形成急剧减少。确定了pAIK - F01和pEdm - F之间存在六个氨基酸差异。直接测序表明，种子株AIK - C的F蛋白第278位氨基酸为亮氨酸（Leu）或苯丙氨酸（Phe）。使用重组F蛋白质粒的实验表明，第278位为亮氨酸的质粒诱导多核体形成能力较差，而第278位为苯丙氨酸（Edmonston型）的质粒诱导广泛的细胞融合。将pEdm - F第278位的苯丙氨酸替换为亮氨酸会降低融合诱导能力。构建了F蛋白第278位为亮氨酸的AIK - C全长感染性克隆。拯救出的病毒在Vero细胞中形成小空斑。然而，相同的拯救病毒若第278位为苯丙氨酸则形成大空斑。得出结论，MV疫苗株AIK - C的F蛋白第278位的亮氨酸导致了小空斑的形成。
