Peluso R W, Richardson J C, Talon J, Lock M
Department of Microbiology, Mt. Sinai School of Medicine, New York, New York 10029, USA.
Virology. 1996 Apr 15;218(2):335-42. doi: 10.1006/viro.1996.0202.
Previous work (C.F. Spiropoulou and S.T. Nichol, 1993, J. Virol. 67, 3103-3110) has demonstrated the existence in cells infected with the New Jersey serotype of vesicular stomatitis virus (VSV) of two small carboxy-coterminal proteins encoded by the P mRNA. These proteins have been named C' and C. We are interested in studying the function of these proteins in the virus life cycle. Toward this end, we have cloned the ORF encoding the potential C' protein of the Indiana serotype as a histidine-tagged fusion protein, purified the expressed protein from Escherichia coli, and used the fusion protein as an immunogen to raise antiserum in a rabbit. We have used this anti-C' protein serum to demonstrate that both of the predicted C' and C proteins are synthesized in cells infected with the Indiana serotype of VSV. In addition we have localized a portion of these proteins to nucleocapsids isolated from infected cells, suggesting that they may play a role in RNA synthesis. Reconstitution of the viral polymerase activity by expressing the L and P protein subunits with or without the C proteins failed to demonstrate any effect of the presence of these latter proteins on reconstituted transcription using purified nucleocapsids as templates. However, we have been able to show a dramatic stimulation of the polymerase activity in purified virions by the addition of purified C' protein to in vitro transcription reactions. Both the level and the fidelity of mRNA synthesis are stimulated by this protein. Evidence for the specificity of this effect comes from the fact that stimulation appears to be serotype specific; C' protein of the Indiana serotype stimulates transcription by purified Indiana serotype virions but has a minimal effect on transcription by purified virions of the New Jersey serotype. We are continuing our studies to determine the mechanism of this stimulation.
先前的研究工作（C.F. Spiropoulou和S.T. Nichol，1993年，《病毒学杂志》67卷，3103 - 3110页）已证明，在感染水疱性口炎病毒（VSV）新泽西血清型的细胞中，存在由P mRNA编码的两种小的羧基末端相同的蛋白质。这些蛋白质已被命名为C'和C。我们有兴趣研究这些蛋白质在病毒生命周期中的功能。为此，我们已克隆了编码印第安纳血清型潜在C'蛋白的开放阅读框（ORF），将其作为组氨酸标签融合蛋白，从大肠杆菌中纯化表达的蛋白，并使用该融合蛋白作为免疫原在兔体内制备抗血清。我们已使用这种抗C'蛋白血清证明，在感染VSV印第安纳血清型的细胞中，预测的C'和C蛋白均会合成。此外，我们已将这些蛋白质的一部分定位到从感染细胞中分离出的核衣壳上，这表明它们可能在RNA合成中发挥作用。通过表达L和P蛋白亚基（有或没有C蛋白）来重建病毒聚合酶活性，未能证明这些后一种蛋白的存在对以纯化核衣壳为模板的重建转录有任何影响。然而，我们已经能够证明，通过在体外转录反应中添加纯化的C'蛋白，可显著刺激纯化病毒粒子中的聚合酶活性。这种蛋白可刺激mRNA合成的水平和保真度。这种效应具有特异性的证据来自以下事实：刺激似乎具有血清型特异性；印第安纳血清型的C'蛋白可刺激纯化的印第安纳血清型病毒粒子的转录，但对新泽西血清型纯化病毒粒子的转录影响极小。我们正在继续研究以确定这种刺激的机制。
