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重大的公共卫生挑战，需要新的治疗方法 策略。 Viperin（病毒抑制蛋白，内质网相关蛋白，干扰素 (IFN) 诱导型）， 自由基 S-腺苷甲硫氨酸 (RS) 酶超家族的成员，是一种干扰素诱导蛋白 抑制多种病毒的复制，包括基孔肯雅病毒、布尼亚姆维拉病毒、 蜱传脑炎病毒、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、HIV 和许多其他 DNA 和 RNA 病毒。蝰蛇已经 建议通过与大量的相互作用或共定位来引发这些深远的抗病毒活性 功能上不相关的宿主和病毒蛋白的数量。所有这些相互作用都是基于间接方法 （例如，酵母双杂交和免疫沉淀），并且没有经过直接生化验证 接近。 viperin 全面抗病毒活性的机制仍然是个谜，尚不清楚 单个蛋白质（即蝰蛇蛋白）如何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 病毒阵列。相反，我们赞成对这些抗病毒活性进行更普遍的机械解释。一 涉及蝰蛇蛋白介导的酶促转化，调节常见的特定细胞过程 对于所有这些病毒。 我们证明，与之前的所有工作相反，viperin 将胞苷三磷酸 (CTP) 转化为 通过 S-腺苷甲硫氨酸 (SAM) 依赖性自由基机制实现新型 CTP 相关三磷酸 与 RS 超家族的其他成员类似。这种新分子的体内功能仍然存在 待定义；但可能包括 1) 病毒 RNA 和 DNA 聚合酶的选择性“中毒”，2) 胞苷酰转移酶的调节/抑制，其使用 CTP 作为底物，并且是脂质所必需的 生物合成（例如磷脂酰乙醇胺、磷脂酰胆碱）和 3) 作为新型信号传导的作用 分子。所有这些可能性将为 viperin 抗病毒功能提供一个统一的机制，因为每个 所提出的机制依赖于蝰蛇蛋白基于自由基的酶特性来调节基本 对所有病毒物种都至关重要的过程（复制、膜动力学和信号传导）。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制 其生产细节。 目标 2：确定 CTP 衍生分子的体内作用。 目标 3：确定蝰蛇蛋白单独、与底物和产物的 X 射线结构。