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.

各种病毒感染继续代表着重大的公共卫生挑战，并要求新疗法 策略。耐蛋白（病毒抑制蛋白，内质网相关，干扰素（IFN）诱导），A 酶的自由基S-腺苷甲硫代（RS）超家族的成员是一种干扰素诱导蛋白 这抑制了包括Chikungunya病毒，Bunyamwera病毒在内的多种病毒的复制 tick出生的脑炎病毒，影响力病毒，人类巨细胞病毒，西尼罗河病毒，丙型肝炎病毒， 信德氏病毒，日本脑炎病毒，HIV以及许多其他DNA和RNA病毒。 viperin曾经 建议通过与大的相互作用或共定位来引起这些深远的抗病毒活性 功能无关的宿主和病毒蛋白的数量。所有这些相互作用均基于间接方法 （例如，酵母2杂交和免疫沉淀），没有直接生化验证 方法。 viperin扫描抗病毒活性的基础机制仍然神秘，还不清楚 单一蛋白（即耐蛋白）如何参与如此广泛的相互作用播放列表以抑制这种宽度 一系列病毒。取而代之的是，我们赞成对这些抗病毒活性的更通用的机械解释。一 涉及耐蛋白介导的酶转化，该酶转化调节特定的细胞过程常见 所有这些病毒。 我们证明，与所有先前的工作形成对比，耐蛋白将三磷酸细胞磷酸酯（CTP）转换为 新型CTP相关的三磷酸通过S-腺苷硫氨酸（SAM）依赖性自由基机理 类似于RS超家族的其他成员。该新分子的体内功能仍然存在 被定义；但可能包括1）病毒RNA和DNA聚合酶的选择性“中毒”，2） 使用CTP作为底物的胞质转移的调节/抑制作用，是脂质所必需的 生物合成（例如，磷脂酰甲醇胺，磷脂酰胆碱）和3）作为一种新的信号传导的作用 分子。所有这些可能性都将为耐毒素抗病毒功能提供统一的机制，因为 提出的机制依赖于基于自由基的酶酶特性来调节基本 对所有病毒规格至关重要的过程（复制，膜动力学和信号传导）。我们的具体目的是： AIM 1：明确定义了新的CTP衍生分子的结构和机械 其生产的细节。 目标2：确定CTP衍生分子的体内作用。 AIM 3：单独使用底物和产物确定耐蛋白蛋白的X射线结构。