Evaluation of SARS-CoV 2'O Methyltransferase Mutants

SARS-CoV 2O 甲基转移酶突变体的评估

• 批准号: 8839128
• 负责人: VINEET D MENACHERY
• 金额: $ 5.51万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8839128
• 关键词: Acute; Adult Respiratory Distress Syndrome; Age; Alveolar; Amino Acid Motifs; Animal Model; Antiviral Agents; Attenuated; Attenuated Vaccines; Body Weight decreased; Coronavirus; Coronavirus Infections; DNA Viruses; Data; Diffuse; Disease Outbreaks; Evaluation; Event; Family; Family member; Genes; Guanine; Human; Human Virus; Immune; Immune response; In Vitro; Infection; Interferon Type I; Interferons; Kinetics; Laboratories; Lung diseases; Mammalian Cell; Methylation; Methyltransferase; Modeling; Morbidity - disease rate; Mus; Nucleosides; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Phase; Play; Process; Proteins; Publishing; RNA; RNA Caps; RNA Viruses; Research; Role; S-Adenosylmethionine; SH2D3A gene; Severe Acute Respiratory Syndrome; Severities; Signal Transduction; Testing; Therapeutic; Time; Vaccination; Vaccine Design; Vaccines; Vertebral column; Viral; Viral Genes; Virulence; Virus; Virus Diseases; Virus Replication; Work; aged; attenuation; atypical pneumonia; base; gene function; in vivo; insight; interest; mortality; mutant; public health relevance; recombinant virus; response; scaffold; small hairpin RNA; success; therapeutic target; transmission process; treatment strategy; viral DNA

DESCRIPTION (provided by applicant): Coronaviruses (CoV) are important emerging human viruses, with multiple cross-species transmission events identified over the past 200 years. Human infections with severe acute respiratory syndrome coronavirus (SARS-CoV), the first newly emerged virus of the 21st century, results in acute and organizing phase diffuse alveolar damage, atypical pneumonia, and acute respiratory distress syndrome (ARDS), leading to mortality rates of 10-50 percent, dependent on age. A major component in the success of the SARS-CoV is its ability to manipulate and subvert the host immune response. Studies by our laboratory and others have revealed numerous genes that antagonize the type I interferon (IFN) response including NSP1, ORF3b, and ORF6. In addition, several groups have generated SARS-CoV deletion mutants and demonstrated various levels of attenuation, possibly due to increased type I IFN sensitivity. A central hypothesis in this application is that highly conserved viral gene functions that target type I IFN responses can serve to provide a universal platform for the rational design of vaccine and drug therapeutics affording rapid responses in outbreak settings. Recently, research has focused on viral components involved in type I RNA capping utilized by SARS, other CoV, and many RNA and DNA viruses. In an ordered process, several CoV proteins contribute to the capping process including NSP13 (RTPase), NSP14 (N7-guanine methylation), and NSP10 (scaffold). However, interest has focused on NSP16, a S-adenosylmethionine (SAM) dependent nucleoside 2'-O-methyltransferase (2'-O-MTase) and its critical role in subverting the type I IFN response. Recent works have implicated 2'O- methylation in distinguishing between self and non-self RNA by MDA-5, a RIG-I like recognition molecule, and the IFIT family of interferon stimulated genes (ISGs). These results suggest that 2'-O-MTases like NSP16 play a critical role in immune antagonism during viral infection. Based on these recent findings, we sought to evaluate the impact of 2'O-methylation on SARS-CoV replication and pathogenesis by generating mutants lacking NSP16 2'O-MTase activity. We hypothesize that deltaNSP16 mutant viruses will be exquisitely sensitive to and attenuated in the presence of type I IFN both in vitro and in vivo. We anticipate the absence of type I IFN signaling or specific ISGs including MDA5 and IFIT family members will restore replication and possibly virulence. Due to broad conservation of this activity across RNA and DNA virus families, targeting the 2'O methylation pathways with either vaccine or drug strategies may provide unique, broadly applicable treatment options that can protect against current and emerging viruses.

描述（由申请人提供）：冠状病毒（CoV）是重要的新兴人类病毒，在过去200年中发现了多种跨物种传播事件。严重急性呼吸综合征冠状病毒（SARS-CoV）是21世纪世纪第一个新出现的病毒，人类感染SARS-CoV会导致急性和机化期弥漫性肺泡损伤、非典型肺炎和急性呼吸窘迫综合征（ARDS），导致10%-50%的死亡率，取决于年龄。SARS-CoV成功的一个主要因素是它能够操纵和破坏宿主的免疫反应。我们实验室和其他人的研究已经揭示了许多拮抗I型干扰素（IFN）反应的基因，包括NSP 1，ORF 3b和ORF 6。此外，几个研究小组已经产生了SARS-CoV缺失突变体，并证明了不同程度的减毒，可能是由于I型IFN敏感性增加。本申请中的一个中心假设是， 靶向I型IFN应答的病毒基因功能可用于为疫苗和药物治疗的合理设计提供通用平台，从而在爆发环境中提供快速应答。最近，研究集中在SARS、其他CoV和许多RNA和DNA病毒所利用的I型RNA加帽中涉及的病毒组分。在一个有序的过程中，几种CoV蛋白有助于加帽过程，包括NSP 13（RTG），NSP 14（N7-鸟嘌呤甲基化）和NSP 10（支架）。然而，兴趣集中在NSP 16，S-腺苷甲硫氨酸（SAM）依赖性核苷2 ′-O-甲基转移酶（2 ′-O-MTase）及其在破坏I型IFN应答中的关键作用。最近的研究表明，2 'O-甲基化通过MDA-5（一种RIG-I样识别分子）和干扰素刺激基因（ISG）的IFIT家族来区分自身和非自身RNA。这些结果表明，2 '-O-MT酶如NSP 16在病毒感染期间的免疫拮抗作用中起关键作用。基于这些最新的发现，我们试图通过产生缺乏NSP 16 2 'O-MTase活性的突变体来评估2' O-甲基化对SARS-CoV复制和致病性的影响。我们假设deltaNSP 16突变病毒将在体外和体内对I型IFN的存在下非常敏感并减毒。我们预期I型IFN信号传导或特异性ISG（包括MDA 5和IFIT家族成员）的缺失将恢复复制和可能的毒力。由于这种活性在RNA和DNA病毒家族中的广泛保守性，用疫苗或药物策略靶向2 'O甲基化途径可以提供独特的、广泛适用的治疗选择，可以保护免受当前和新出现的病毒的侵害。