Function and Mechanism of Viperin, a radical SAM antiviral protein

各种病毒感染仍然是公共卫生的重大挑战，需要新的治疗方法 战略。Viperin(病毒抑制蛋白，内质网相关，干扰素(干扰素)诱导)，a S-腺苷蛋氨酸(RS)超家族成员是一种干扰素诱导蛋白 这抑制了一系列病毒的复制，包括基孔肯雅病毒、本扬维拉病毒、 森林脑炎病毒、甲型流感病毒、人类巨细胞病毒、西尼罗河病毒、丙型肝炎病毒、 辛德比斯病毒、日本脑炎病毒、艾滋病毒和许多其他DNA和RNA病毒。毒蛇已经被 建议通过与大型企业的互动或共同本地化来激发这些影响深远的抗病毒活动 功能无关的宿主和病毒蛋白的数量。所有这些交互都是基于间接方法的 (例如，酵母双杂交和免疫沉淀)，没有一个是通过直接生化验证的 接近了。毒蛇毒素广泛的抗病毒活性背后的机制仍然是个谜，目前还不清楚 单一蛋白质(即毒蛇蛋白)如何参与如此广泛的相互作用以抑制这种广泛的 一系列病毒。相反，我们倾向于对这些抗病毒活动进行更一般的机制解释；一 这涉及到毒蛇毒素介导的酶转化，它调节常见的特定细胞过程 所有这些病毒。 我们证明，与所有以前的工作相反，毒蛇毒素将胞苷三磷酸(CTP)转化为 通过S-腺苷甲硫氨酸依赖的自由基机制实现CTP相关的新三磷酸 类似于RS超家族的其他成员。这种新分子的体内功能仍然存在 有待定义；但可能包括1)选择性的病毒RNA和DNA聚合酶“中毒”，2) 胞苷酰转移酶的调节/抑制，它使用CTP作为底物，是脂质所必需的 生物合成(如磷脂酰乙醇胺、磷脂酰胆碱)和3)作为一种新信号的作用 分子。所有这些可能性都将为毒蛇毒素的抗病毒功能提供统一的机制，因为每一种 所提出的机制依赖于基于自由基的蛇毒的酶性质来调节基础 对所有病毒物种至关重要的过程(复制、膜动力学和信号传递)。我们的具体目标是： 目标1：明确定义新的CTP衍生分子的结构和机理 它的生产细节。 目的2：确定CTP衍生分子在体内的作用。 目的3：测定毒蛇抗菌素单独、底物和产物的X射线结构。

Viral infections of all kinds continue to represent major public health challenges and demand new therapeutic strategies. Viperin (virus-inhibitory protein, endoplasmic reticulum associated, interferon (IFN) inducible), a member of the radical S-adenosylmethionine (RS) superfamily of enzymes, is an interferon inducible protein that inhibits the replication of a remarkable range of viruses, including Chikungunya virus, Bunyamwera virus, Tick-born encephalitis virus, influenza A virus, human cytomegalovirus, West Nile virus, hepatitis C virus, sindbis virus, Japanese encephalitis virus, HIV and numerous other DNA and RNA viruses. Viperin has been suggested to elicit these far-reaching antiviral activities through interaction or co-localization with a large number of functionally unrelated host and viral proteins. All of these interactions are based on indirect methods (e.g., yeast-two-hybrid and immunoprecipitation), and none have been validated by direct biochemical approaches. The mechanisms underlying viperin’s sweeping antiviral activity remain enigmatic and it is unclear how a single protein (i.e., viperin) can participate in such a broad playlist of interactions to inhibit this wide array of viruses. Instead, we favor a more general mechanistic explanation for these antiviral activities; one that involves a viperin-mediated enzymatic transformation that modulates specific cellular processes common to all of these viruses. We demonstrate that, contrary to all previous work, viperin converts cytidine triphosphate (CTP) to a novel CTP-related triphosphate via an S-adenosylmethionine (SAM)-dependent radical mechanism analogous to other members of the RS superfamily. The in vivo function of this new molecules remains to be defined; but may include 1) selective “poisoning” of viral RNA and DNA polymerases, 2) modulation/inhibition of cytidylyl transferases, which use CTP as a substrate, and are required for lipid biosynthesis (e.g., phosphatidylethanolamine, phosphatidylcholine) and 3) a role as a novel signaling molecule. All of these possibilities would provide a unified mechanism for viperin antiviral function, as each proposed mechanism relies on the radical-based enzymatic properties of viperin to modulate fundamental processes (replication, membrane dynamics and signaling) critical to all viral species. Our Specific Aims are: Aim 1: Unambiguously define the structure of the new CTP-derived molecule and the mechanistic details of its production. Aim 2: Determine the in vivo role of the CTP-derived molecule. Aim 3: Determine the X-ray structures of viperin alone, with substrate and with product.