Meier E, Harmison G G, Schubert M
J Virol. 1987 Oct;61(10):3133-42. doi: 10.1128/JVI.61.10.3133-3142.1987.
The recombinant polymerase protein L of vesicular stomatitis virus (VSV) expressed in COS cells is able to transcribe and replicate the viral genome, resulting in complementation of temperature-sensitive polymerase mutants of VSV at the restrictive temperature (M. Schubert, G. G. Harmison, C. D. Richardson, and E. Meier, Proc. Natl. Acad. Sci. USA 82:7984-7988, 1985). Here we report that the efficiency of complementation is dependent on the level of L protein expression. Unexpectedly, only cells expressing low levels of recombinant L protein efficiently complemented tsL gene mutants, whereas cells with high levels of L protein did not. In fact, in all cells with high levels of L protein expression, which at 40 h posttransfection represented almost the total number of transfected cells, viral replication not only of the temperature-sensitive mutant but also of wild-type VSV was excluded. The inhibition of VSV appeared to occur at an early stage of the infectious cycle, and wild-type virus of the same serotype (Indiana) as the recombinant L protein as well as wild-type virus of a different serotype (New Jersey) was affected. Measles virus, on the other hand, was not arrested in cells with high levels of recombinant L protein, demonstrating that these cells were still capable of supporting a viral infection. The expression of high levels of only the amino-terminal half of the L protein from a recombinant mutant L gene that contains a small out-of-frame deletion in the middle of the L gene did not inhibit a VSV infection. Since the level of amplification for both L- and truncated L-encoding vectors is similar, we conclude that the arrest of VSV was caused by high levels of functional full-length L protein itself and not by high levels of vector-encoded L mRNA or other vector products or by side effects of vector amplification. These data strongly support the idea that the highly conserved gene order of nonsegmented negative-strand viruses and the sequential and attenuated mode of transcription are important regulatory elements which balance the intracellular concentration of viral proteins. They both assure that the L gene is the last and the least frequently transcribed gene, giving rise to low levels of L protein necessary for efficient replication.
在COS细胞中表达的水疱性口炎病毒（VSV）重组聚合酶蛋白L能够转录和复制病毒基因组，从而在限制温度下对VSV的温度敏感型聚合酶突变体进行互补（M. 舒伯特、G. G. 哈米森、C. D. 理查森和E. 迈尔，《美国国家科学院院刊》82:7984 - 7988, 1985）。在此我们报告，互补效率取决于L蛋白的表达水平。出乎意料的是，只有表达低水平重组L蛋白的细胞能有效地互补tsL基因突变体，而L蛋白水平高的细胞则不能。实际上，在所有转染后40小时表达高水平L蛋白的细胞中（这些细胞几乎占转染细胞的总数），不仅温度敏感型突变体的病毒复制被排除，野生型VSV的病毒复制也被排除。VSV的抑制似乎发生在感染周期的早期阶段，与重组L蛋白同血清型（印第安纳型）的野生型病毒以及不同血清型（新泽西型）的野生型病毒均受到影响。另一方面，麻疹病毒在表达高水平重组L蛋白的细胞中并未被阻断，这表明这些细胞仍然能够支持病毒感染。来自重组突变L基因（该基因在L基因中部有一个小的移码缺失）的仅L蛋白氨基末端一半的高水平表达并未抑制VSV感染。由于编码L蛋白和截短L蛋白的载体的扩增水平相似，我们得出结论，VSV的阻断是由高水平的功能性全长L蛋白本身引起的，而不是由高水平的载体编码L mRNA或其他载体产物或载体扩增的副作用引起的。这些数据有力地支持了这样一种观点，即非节段性负链病毒高度保守的基因顺序以及转录的顺序和衰减模式是重要的调节元件，它们平衡了病毒蛋白在细胞内的浓度。它们都确保L基因是最后且转录频率最低的基因，从而产生高效复制所需的低水平L蛋白。
