Mechanism of action of an African swine fever virus virulence factor

非洲猪瘟病毒（ASFV）是一种具有高死亡率的经济上重要的猪疾病。我们计划研究由病毒编码的蛋白质之一（DP71L），以确定其在细胞中的功能及其在引起猪疾病中的作用。先前的研究表明，删除这种蛋白质的基因可以降低ASFV在猪中的毒力。这种蛋白质在其部分长度上与单纯疱疹病毒毒力因子ICP 34.5和由诸如DNA损伤的因素诱导的宿主蛋白质（GADD 34）相似。我们最近已经表明，DP71L具有ICP34.5或GADD 34未证明的两种新功能。首先，它干扰干扰素（IFN）的诱导，这激活了一个重要的宿主抗病毒途径。它这样做的机制尚不清楚。其次，DP71L也通过未知的机制刺激细胞酶蛋白磷酸酶1（PP1）。PP1可以通过与调节蛋白结合来激活或抑制其他细胞途径。我们建议DP71L蛋白作为PP1的调节剂，以取代PP1的抑制剂，并靶向酶来调节宿主细胞途径的活性。这可能是DP71L抑制IFN诱导的机制。我们的预测是DP71L可能抑制其他细胞功能。DP71L蛋白的突变形式将被制备并用于研究蛋白的哪些部分是其每种功能所需的。我们还将确定DP71L在哪个点阻断IFN诱导途径，并研究DP71L是否影响其他宿主途径。特别是，我们将研究是否DP71L影响其他细胞基因的表达，除了IFN，这是重要的刺激宿主的感染反应。我们还将研究DP71L是否阻断限制病毒复制的其他两种宿主途径。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基因的缺失显著降低了猪的毒力。通过制备表达突变形式的DP71L蛋白的重组病毒，我们将确定是否需要该蛋白的任一或两种功能来获得毒力。我们还将确定我们在分离的细胞中观察到的功能是否也在感染的猪中观察到。这些结果将有助于我们更多地了解IFN的诱导机制，以及PP1的功能，以及病毒如何引导IFN以避免宿主的防御。了解单个蛋白在毒力中的作用将有助于合理开发ASFV减毒疫苗。目前没有疫苗可用，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.