Black T L, Barber G N, Katze M G
Department of Microbiology, School of Medicine, University of Washington, Seattle 98195.
J Virol. 1993 Feb;67(2):791-800. doi: 10.1128/JVI.67.2.791-800.1993.
Control of the interferon-induced double-stranded RNA (dsRNA) activated protein kinase (referred to as P68 because of its M(r) of 68,000 in human cells) by animal viruses is essential to avoid decreases in protein synthetic rates during infection. We have previously demonstrated that poliovirus establishes a unique way of regulating the protein kinase, namely by inducing the specific degradation of P68 during infection (T. L. Black, B. Safer, A. Hovanessian, and M. G. Katze, J. Virol. 63:2244-2251, 1989). In the present study we investigated the mechanisms by which P68 degradation occurred. To do this we used an in vitro degradation assay which faithfully reproduced the in vivo events. Although viral gene expression was required for P68 degradation, the major poliovirus proteases, 2A and 3C, were found not to be directly involved with P68 proteolysis. However, the protease responsible for P68 degradation required divalent cations for maximal activity and probably has both an RNA and a protein component since trypsin and ribonuclease abrogated the activity. Despite this requirement for divalent cations and RNA, activation of the kinase was not required for proteolysis since a catalytically inactive P68 was still degraded. Mapping of P68 protease-sensitive sites by using in vitro translated truncation and deletion mutants revealed that sites required for degradation resided in the amino terminus and colocalized to dsRNA-binding domains. Finally, we found that preincubation of cell extracts with the synthetic dsRNA poly(I-C) largely prevented P68 proteolysis, providing additional evidence for the critical role of RNA. On the basis of these data, we present a hypothetical model depicting possible mechanisms of P68 degradation in poliovirus-infected cells.
动物病毒对干扰素诱导的双链RNA（dsRNA）激活蛋白激酶（在人类细胞中因其分子量为68,000而被称为P68）的控制对于避免感染期间蛋白质合成速率的降低至关重要。我们之前已经证明，脊髓灰质炎病毒建立了一种独特的调节该蛋白激酶的方式，即在感染期间诱导P68的特异性降解（T. L. Black、B. Safer、A. Hovanessian和M. G. Katze，《病毒学杂志》63:2244 - 2251，1989）。在本研究中，我们研究了P68降解发生的机制。为此，我们使用了一种体外降解试验，该试验忠实地再现了体内事件。虽然P68降解需要病毒基因表达，但发现脊髓灰质炎病毒的主要蛋白酶2A和3C并不直接参与P68的蛋白水解。然而，负责P68降解的蛋白酶需要二价阳离子以达到最大活性，并且可能同时具有RNA和蛋白质成分，因为胰蛋白酶和核糖核酸酶会消除其活性。尽管需要二价阳离子和RNA，但蛋白水解并不需要激酶的激活，因为催化无活性的P68仍然会被降解。通过使用体外翻译的截短和缺失突变体对P68蛋白酶敏感位点进行定位，发现降解所需的位点位于氨基末端，并与dsRNA结合域共定位。最后，我们发现用合成的dsRNA聚肌胞苷酸（poly(I-C)）预孵育细胞提取物可在很大程度上防止P68蛋白水解，这为RNA的关键作用提供了额外证据。基于这些数据，我们提出了一个假设模型，描述了脊髓灰质炎病毒感染细胞中P68降解的可能机制。
