Function and Mechanism of Viperin, a radical SAM antiviral protein

自由基SAM抗病毒蛋白Viperin的功能和机制

• 批准号: 9375148
• 负责人: STEVEN C. ALMO
• 金额: $ 25.05万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-26 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9375148
• 关键词: Antiviral Agents; Biochemical; Biological; Biology; Bunyamwera virus; Catalytic Domain; Cell physiology; Cells; Chemistry; Chikungunya virus; Collaborations; Complex; Crystallization; Cytidine; Cytomegalovirus; DNA Viruses; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Development; Encephalitis Viruses; Endoplasmic Reticulum; Enzymes; Exploratory/Developmental Grant; Future Generations; Genetic Transcription; HIV; Hepatitis C; Human; Immunoprecipitation; In Vitro; Influenza A virus; Interferons; Isotope Labeling; Japanese encephalitis virus; Lecithin; Ligands; Lipids; Mediating; Membrane; Methods; Phosphatidylethanolamine; Poisoning; Polymerase; Positioning Attribute; Process; Production; Property; Proteins; Public Health; RNA Viruses; Reaction; Resolution; Roentgen Rays; Role; S-Adenosylmethionine; Signal Pathway; Signal Transduction; Signaling Molecule; Sindbis Virus; Site; Spectrum Analysis; Structure; Testing; Ticks; Transferase; Viral; Viral Proteins; Virus; Virus Diseases; West Nile virus; Work; Yeasts; analog; base; experimental study; high risk; improved; in vivo; influenzavirus; insight; lipid biosynthesis; member; novel; novel therapeutic intervention; programs; transcriptome sequencing; tripolyphosphate; viperin; viral RNA; yeast two hybrid system

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.

各种病毒感染仍然是主要的公共卫生挑战，需要新的治疗方法。 战略布局蝰蛇蛋白（病毒抑制蛋白，内质网相关，干扰素（IFN）诱导）， 自由基S-腺苷甲硫氨酸（RS）超家族的成员，是一种干扰素诱导蛋白 它能抑制一系列病毒的复制，包括基孔肯雅病毒，布尼亚维拉病毒， 蜱传脑炎病毒，甲型流感病毒，人巨细胞病毒，西尼罗河病毒，丙型肝炎病毒， 辛德毕斯病毒、日本脑炎病毒、HIV和许多其它DNA和RNA病毒。蝰蛇一直是 建议通过与大分子的相互作用或共定位来引发这些深远的抗病毒活性。 许多功能上不相关的宿主和病毒蛋白。所有这些相互作用都是基于间接的方法 (e.g.,酵母双杂交和免疫沉淀），并且没有一个被直接生物化学验证。 接近。蝰蛇蛋白广泛的抗病毒活性的机制仍然是个谜， 单一蛋白质（即，蝰蛇素）可以参与如此广泛的相互作用播放列表，以抑制这种广泛的相互作用。 一系列病毒。相反，我们倾向于对这些抗病毒活性进行更普遍的机制解释; 它涉及一种蝰蛇蛋白介导的酶促转化， 所有这些病毒。 我们证明，与所有以前的工作相反，蝰蛇蛋白将三磷酸胞苷（CTP）转化为一种 通过S-腺苷甲硫氨酸（SAM）依赖性自由基机制的新型CTP相关三磷酸 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 但可能包括1）病毒RNA和DNA聚合酶的选择性“中毒”，2） 调节/抑制胞苷酰转移酶，其使用CTP作为底物，并且是脂质合成所需的。 生物合成（例如，磷脂酰乙醇胺，磷脂酰胆碱）和3）作为一种新的信号传导的作用 分子。所有这些可能性将为蝰蛇蛋白的抗病毒功能提供一个统一的机制， 所提出的机制依赖于蝰蛇蛋白的基于自由基的酶性质来调节基本的 这些过程（复制、膜动力学和信号传导）对所有病毒物种至关重要。我们的具体目标是： 目的1：明确定义新CTP衍生分子的结构和机制 生产的细节。 目的2：确定CTP衍生分子的体内作用。 目的3：测定蝰蛇蛋白单独、与底物及与产物的X-射线结构。