Role of RNA Methylation in Regulating HIV Proviral Expression

RNA 甲基化在调节 HIV 原病毒表达中的作用

• 批准号: 10426418
• 负责人: Jian Zhu
• 金额: $ 37.38万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10426418
• 关键词: 3' Untranslated Regions; AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Affect; Anti-Retroviral Agents; Antiviral Agents; Binding; Biological Assay; CD4 Positive T Lymphocytes; Catalytic Domain; Cell Line; Cells; Cytosine; DNA Polymerase II; Distant; Enzymes; Event; Family; Family member; Gene Expression; Genetic; Genetic Transcription; Genetic Translation; HIV; HIV Infections; HIV tat Protein; Highly Active Antiretroviral Therapy; Infection; Interruption; Investigation; Link; Measures; Mediating; Messenger RNA; Methylation; Methyltransferase; Modification; Open Reading Frames; Patients; Play; Positive Transcriptional Elongation Factor B; Proteins; Proviruses; RNA; RNA methylation; Regimen; Regulation; Residual state; Resolution; Rest; Reverse Transcription; Role; Site; Testing; Transcript; Transcriptional Regulation; Translations; Viral Load result; Viral Physiology; Viremia; Virus Latency; antiretroviral therapy; digital; epitranscriptomics; functional genomics; gain of function; improved; innovation; latent HIV reservoir; latent infection; mRNA Stability; member; memory CD4 T lymphocyte; mutant; novel; prevent; reactivation from latency; screening; tat Protein

PROJECT SUMMARY Although HAART treatment is successful to block active replication of HIV in AIDS patients, it does not completely eradicate the infection. HIV latent reservoirs remain as a major obstacle for complete elimination of HIV viruses and cure of the infection. Investigation of host machineries that regulate HIV proviral expression will help to improve the understanding of the stage of HIV latent infection. It will also provide new strategies to perturb host regulatory factors for eliminating latent HIV. We characterized that one of NSUN RNA m5C methyltransferases (m5C-MTases), NSUN1/NOP2, restricts HIV replication, suppresses HIV proviral expression, and promotes viral latency. The impact of m5C methylation catalyzed by NSUN m5C-MTases (NSUN1-7) on HIV replication still remains largely unknown but starts to unfold. In this proposal, we will initiate the in-depth examination of NSUN m5C-MTases as regulators of HIV proviral expression. For aim 1, we will employ the ultra-sensitive reverse transcription droplet digital PCR (RT-ddPCR) assays to measure various HIV transcripts in cells depleted of NSUN m5C-MTases, which will provide a high-resolution profiling of their effect on HIV proviral expression. Furthermore, we will confirm whether the enzymatic activity of NSUN m5C- MTases is required. We will also determine whether NSUN m5C-MTases interferes with HIV post- transcriptional events, including HIV mRNA stability and translation. For aim 2, we will investigate the impact of NSUN m5C-MTases on the activation of P-TEFb and RNA Pol-II that play a critical role in promoting HIV transcription. Our results showed that the loss of NSUN1 reduces m5C methylation of HIV TAR RNA and that its MTase catalytic domain (MTD) prevents HIV Tat-TAR interaction, indicating that m5C methylation of TAR may regulate its interaction with Tat directly. We will identify the m5C methylation site(s) of TAR catalyzed by NSUN m5C-MTases and further determine its role in modulating Tat-TAR interaction. For aim 3, we will investigate the role of NSUN m5C-MTases in regulation of HIV 5’ and 3’ UTRs’ viral functions. We will determine whether NSUN m5C-MTases bind with HIV 5’ and 3’ UTRs as well as contribute to their m5C methylation. Since 5’ and 3’ UTRs of HIV mRNA play a critical role in regulation of HIV proviral expression epi- transcriptionally, we will determine whether m5C methylation of HIV 5’ and 3’ UTRs affects mRNA stability and protein translation. Furthermore, it has been recently shown that NSUN m5C-MTases also play an important role in regulating host cellular epi-transcriptomics. Thus, we will determine their functional impact on host gene expression in HIV latently infected CD4+ T cells. Overall, this proposal will comprehensively investigate NSUN m5C-MTases as novel regulators of HIV proviral expression. We believe that these studies will significantly improve our understanding of host-HIV interactions. From these studies, we will confirm whether members of NSUN m5C-MTases can be targeted for eliminating latent HIV.

项目总结 尽管HAART疗法成功地阻止了艾滋病患者中艾滋病毒的活跃复制，但它并没有 彻底根除感染。艾滋病毒潜伏宿主仍然是彻底消除艾滋病的主要障碍。 HIV病毒和感染的治愈。调节HIV前病毒表达的宿主机制的研究 将有助于提高对HIV潜伏感染阶段的认识。它还将提供新的战略，以 扰乱宿主调节因素以消除潜伏的艾滋病毒。我们鉴定了一种NSUN RNA M5C 甲基转移酶(M5C-MTase)，NSUN1/NOP2，限制HIV复制，抑制HIV前病毒 表达，并促进病毒潜伏期。NSUN M5C-MTase对M5C甲基化的影响 关于艾滋病毒复制的(NSUN1-7)在很大程度上仍不清楚，但已开始展开。在这项提案中，我们将发起 NSUN M5C-MTase作为HIV前病毒表达调节因子的深入研究。对于目标1，我们将 采用超灵敏的反转录液滴数字聚合酶链式反应(RT-ddPCR)检测 NSUN M5C-MTase缺失的细胞中的HIV转录本，这将为他们的 对HIV前病毒表达的影响。此外，我们还将确认NSUN M5C的酶活性是否- MTASS是必需的。我们还将确定NSUN M5C-MTase是否干扰HIV感染后 转录事件，包括HIV信使核糖核酸的稳定性和翻译。对于目标2，我们将调查 NSUN M5C-MTase对促进HIV关键作用的P-TEFb和RNA Pol-II激活的影响 抄写。我们的结果表明，NSUN1的缺失减少了HIV TAR RNA的M5C甲基化，并且 其MTase催化结构域(MTD)阻止HIV TAT-TAR相互作用，表明TAR的M5C甲基化 可能直接调节其与TAT的相互作用。我们将确定焦油的M5C甲基化位点(S)是由 NSUN M5C-MTase，并进一步确定其在调节TAT-TAR相互作用中的作用。对于目标3，我们将 研究NSUN M5C-MTase在HIV 5‘和3’UTRs病毒功能调节中的作用。我们会 确定NSUN M5C-MTase是否与HIV 5‘和3’UTRs结合并促进其M5C 甲基化。由于HIV信使核糖核酸的5‘和3’非编码区在HIV前病毒表达的调控中起着关键作用，因此，HIVmRNA5‘和3’UTRs在HIV前病毒表达的调控中起着重要作用。 在转录方面，我们将确定HIV 5‘和3’UTRs的M5C甲基化是否影响mRNA的稳定性和 蛋白质翻译。此外，最近的研究表明，NSUN M5C-MTase也发挥着重要的作用 在调节宿主细胞表观转录中的作用。因此，我们将确定它们对宿主基因的功能影响 HIV潜伏感染的CD4+T细胞的表达。总体而言，这项提案将全面调查NSUN M5C-MTase作为HIV前病毒表达的新调节因子。我们相信，这些研究将显著地 提高我们对宿主与艾滋病毒相互作用的理解。从这些研究中，我们将确定成员是否 NSUN M5C-MTase可以靶向消除潜伏的HIV。