Blum H, Gross H J, Beier H
Institut für Biochemie, Bayerische Julius-Maximilians-Universität, Würzburg, Federal Republic of Germany.
Virology. 1989 Mar;169(1):51-61. doi: 10.1016/0042-6822(89)90040-8.
The TMV-encoded 30-kDa protein has been implicated in the cell-to-cell transport of TMV in the infected plant. The polyethylene glycol-mediated inoculation of tobacco protoplasts with TMV particles and TMV RNA was used to compare the time courses of the viral 30-kDa protein synthesis in vivo. Upon infection of protoplasts with TMV RNA, the synthesis of the viral 30-kDa protein starts after 4 to 6 hr, has its maximum after 8 to 10 hr, and decreases. After inoculation of protoplasts with TMV, however, the start of the viral 30-kDa protein synthesis and its maximum are delayed by 2 hr, followed by the same decrease. We show that actinomycin D dramatically stimulates the synthesis of the 30-kDa protein by up to 2 orders of magnitude, whereas the synthesis of the viral 126 kDa, the 183 kDa, and the coat protein is increased only by a factor of 2. Surprisingly, actinomycin V is twice as active as actinomycin D, whereas actinomycin I is nearly inactive. The specific stimulation of the 30-kDa synthesis by actinomycin D in vivo depends neither on the Nicotiana variety nor on the TMV strain used. Final evidence that the 30-kDa protein is truly TMV-derived is provided by the slightly different electrophoretic mobilities of the 30-kDa proteins encoded by TMV strains vulgare, dahlemense, and U2. The identification of the 30-kDa protein in two-dimensional gels was achieved for the first time by a combination of ionic and nonionic detergents for the solubilization of the 30-kDa protein and by the specific stimulation of its synthesis by actinomycin D. The mechanism of the strong and selective actinomycin effect on the viral 30-kDa protein synthesis in vivo is as yet obscure. Actinomycin does not appear to act directly on viral protein biosynthesis, since it neither stimulates the 30-kDa synthesis upon translation of TMV RNA in vitro nor alters the ratio of the products. Actinomycin may rather act by inhibiting selectively the synthesis of a host factor whose synthesis starts at least 4 hr after TMV infection and which strongly inhibits the expression of the viral 30-kDa transport protein.
烟草花叶病毒（TMV）编码的30 kDa蛋白与受感染植物中TMV的细胞间运输有关。采用聚乙二醇介导的方法，用TMV颗粒和TMV RNA接种烟草原生质体，以比较体内病毒30 kDa蛋白合成的时间进程。用TMV RNA感染原生质体后，病毒30 kDa蛋白的合成在4至6小时后开始，在8至10小时后达到最大值，然后下降。然而，用TMV接种原生质体后，病毒30 kDa蛋白合成的开始及其最大值延迟2小时，随后同样下降。我们发现放线菌素D可将30 kDa蛋白的合成显著刺激高达2个数量级，而病毒126 kDa、183 kDa蛋白和外壳蛋白的合成仅增加2倍。令人惊讶的是，放线菌素V的活性是放线菌素D的两倍，而放线菌素I几乎无活性。放线菌素D在体内对30 kDa合成的特异性刺激既不依赖于烟草品种，也不依赖于所用的TMV毒株。TMV普通株、达勒姆株和U2株编码的30 kDa蛋白电泳迁移率略有不同，这为30 kDa蛋白确实来源于TMV提供了最终证据。首次通过离子和非离子去污剂组合溶解30 kDa蛋白，并通过放线菌素D对其合成的特异性刺激，在二维凝胶中鉴定出30 kDa蛋白。放线菌素在体内对病毒30 kDa蛋白合成产生强烈且选择性作用的机制尚不清楚。放线菌素似乎不直接作用于病毒蛋白生物合成，因为它在体外翻译TMV RNA时既不刺激30 kDa蛋白的合成，也不改变产物比例。放线菌素可能是通过选择性抑制一种宿主因子的合成来发挥作用，这种宿主因子的合成在TMV感染后至少4小时开始，并且强烈抑制病毒30 kDa运输蛋白的表达。
