Mechanism of action of an African swine fever virus virulence factor

非洲猪瘟病毒(ASFV)是猪的一种重要的经济疾病，死亡率很高。我们计划研究病毒编码的一种蛋白质(DP71L)，以确定它在细胞中的功能以及它在导致猪疾病中所起的作用。先前的工作表明，删除这种蛋白的基因可以降低ASFV对猪的毒力。该蛋白在部分长度上类似于单纯疱疹病毒毒力因子ICP34.5，以及由DNA损伤等因素诱导的宿主蛋白(Gadd34)。我们最近已经证明，DP71L有两个ICP34.5或Gadd34没有展示的新功能。首先，它干扰干扰素的诱导，干扰素激活了宿主抗病毒的一个重要途径。它是如何做到这一点的机制尚不清楚。其次，DP71L刺激一种细胞酶，蛋白磷酸酶1(PP1)，也是通过一种未知的机制。PP1可以通过与调节蛋白结合来激活或抑制其他细胞通路。我们认为DP71L蛋白作为PP1的调节器来取代PP1的抑制剂，并以该酶为靶点来调节宿主细胞通路的活性。这可能是DP71L抑制干扰素诱导的机制之一。我们的预测是，DP71L可能会抑制其他细胞功能。将制造DP71L蛋白的突变形式，并用于研究其每种功能所需的蛋白质部分。我们还将确定DP71L在哪个点阻断干扰素诱导途径，并调查DP71L是否影响其他宿主途径。特别是，我们将研究DP71L是否影响除干扰素之外的其他细胞基因的表达，这些基因对于刺激宿主对感染的反应是重要的。我们还将研究DP71L是否阻止了另外两条限制病毒复制的宿主途径。在这些途径中，宿主细胞要么自杀(称为凋亡)，要么关闭信使RNA的翻译，以防止病毒复制。DP71L基因的缺失大大降低了猪的毒力。通过制造表达突变形式的DP71L蛋白的重组病毒，我们将确定该蛋白的其中一种功能或两种功能是毒力所必需的。我们还将确定我们在分离细胞中观察到的功能是否也在感染猪身上观察到。这些结果将有助于我们更多地了解诱导干扰素的机制，以及PP1的功能，以及病毒如何引导这一功能来避开宿主的防御。了解单个蛋白在毒力中的作用将有助于合理开发减毒ASFV疫苗。目前还没有疫苗可用，因此疾病控制依赖于快速诊断和实施检疫和屠宰。

African swine fever virus (ASFV) causes an economically important disease of pigs with high mortality rates. We plan to study one of the proteins (DP71L) encoded by the virus to determine its function in cells and the role it plays in causing disease in pigs. Previous work has shown that deleting the gene for this protein can reduce ASFV virulence in pigs. This protein is similar over part of its length to a Herpes simplex virus virulence factor, ICP34.5, and to a host protein (GADD34) which is induced by factors such as damage to DNA. We have recently shown that DP71L has two novel functions not demonstrated for ICP34.5 or GADD34. First it interferes with induction of interferon (IFN), which activates an important host anti-viral pathway. The mechanism by which it does this is not known. Secondly, DP71L stimulates a cellular enzyme, protein phosphatase 1 (PP1), also by an unknown mechanism. PP1 can be directed to activate or inhibit other cellular pathways by association with regulatory proteins. We propose that the DP71L protein acts as a regulator of PP1 to displace an inhibitor from PP1 and target the enzyme to modulate the activity of host cell pathways. This may be the mechanism by which DP71L inhibits induction of IFN. Our prediction is that DP71L may inhibit other cellular functions. Mutant forms of the DP71L protein will be made and used to study which parts of the protein are needed for each of its functions. We will also determine at which point DP71L blocks the IFN induction pathway and investigate if DP71L affects other host pathways. In particular we will study whether DP71L affects expression of other cellular genes, in addition to IFN, which are important for stimulating the host's response to infection. We will also study whether DP71L blocks two other host pathways which restrict virus replication. In these pathways the host cell either commits suicide (called apoptosis) or shuts-off translation of messenger RNAs to prevent virus from replicating. Deletion of the DP71L gene dramatically reduces virulence in pigs. By making recombinant virus which expresses mutant forms of the DP71L protein, we will determine if either or both functions of the protein are required for virulence. We will also determine if the functions we observe in isolated cells are also observed in infected pigs. The results will help us to understand more about the mechanisms by which IFN is induced, and the functions of PP1 and how this may be directed by viruses to avoid the host's defences. Understanding the role of individual proteins in virulence will assist in the rational development of an attenuated ASFV vaccine. Currently no vaccine is available so disease control relies on rapid diagnosis and implementation of quarantine and slaughter.