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.

项目摘要 尽管HAART治疗成功地阻止了艾滋病患者的艾滋病毒的主动复制，但是 完全消除感染。艾滋病毒潜伏水库仍然是完全消除的主要障碍 HIV病毒和感染的治愈。调查调节HIV病毒表达的宿主机器 将有助于提高对艾滋病毒潜伏感染阶段的理解。它还将提供新的策略 扰动宿主调节因素，以消除潜在的艾滋病毒。我们表征了NSUN RNA M5C之一 甲基转移酶（M5C-MTases），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-MTases催化的M5C甲基化的影响 （NSUN1-7）关于艾滋病毒的复制，仍然在很大程度上未知，但开始展开。在此提案中，我们将发起 对NSUN M5C-MTases的深入研究是HIV临时表达的调节剂。对于目标1，我们将 使用超敏感的逆转录液滴数字PCR（RT-DDPCR）测定测量 NSUN M5C-MTases耗尽的细胞中的HIV转录本将提供其高分辨率分析 对HIV病毒表达的影响。此外，我们将确认NSUN M5C-的酶活性是否是 需要mtases。我们还将确定NSUN M5C-MTases是否干扰HIV后 转录事件，包括HIV mRNA稳定性和翻译。对于AIM 2，我们将调查 NSUN M5C-MTases关于P-TEFB和RNA POL-II的激活，在促进HIV中起着关键作用 转录。我们的结果表明，NSUN1的损失降低了HIV TAR RNA的M5C甲基化，并且 其MTase催化结构域（MTD）可防止HIV TAT-TAT相互作用，表明M5C的M5C甲基化 可以直接调节其与TAT的相互作用。我们将确定由TAR催化的M5C甲基化位点 NSUN M5C-MTases并进一步确定了其在调节TAT-TAR相互作用中的作用。对于目标3，我们将 研究NSUN M5C-MTases在HIV 5'和3'UTRS病毒功能的调节中的作用。我们将 确定NSUN M5C-MTases是否与HIV 5'和3'UTR结合并有助于其M5C 甲基化。由于HIV mRNA的5'和3'UTR在调节HIV病毒表达表演中起关键作用 在转录上，我们将确定HIV 5'和3'UTR的M5C甲基化是否会影响mRNA稳定性和 蛋白质翻译。此外，最近已经表明，NSUN M5C-MTases也起着重要的作用 控制宿主细胞表周期转录组学中的作用。这，我们将确定它们对宿主基因的功能影响 在HIV中的表达潜在地感染了CD4+ T细胞。总体而言，该建议将全面调查NSUN M5C-MTases是HIV临时表达的新型调节剂。我们认为这些研究将大大 提高我们对宿主-HIV互动的理解。从这些研究中，我们将确认是否成员 NSUN M5C-MTases可以针对消除潜在的HIV。