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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

项目概要尽管HAART治疗成功地阻止了艾滋病患者体内HIV的活跃复制，但它并不能彻底消除艾滋病毒潜伏病毒库仍然是彻底消除艾滋病毒的主要障碍。 HIV病毒和感染的治愈的研究调节HIV原病毒表达的宿主机器。将有助于提高对HIV潜伏感染阶段的认识，也将为研究提供新的策略。我们鉴定了 NSUN RNA m5C 之一。甲基转移酶 (m5C-MTases)、NSUN1/NOP2，限制 HIV 复制，抑制 HIV 原病毒 NSUN m5C-MTase 催化的 m5C 甲基化的影响。 (NSUN1-7) 关于 HIV 复制的情况仍然很大程度上未知，但在本提案中我们将开始展开。深入研究 NSUN m5C-MTase 作为 HIV 原病毒表达的调节剂对于目标 1，我们将采用超灵敏逆转录液滴数字 PCR (RT-ddPCR) 测定法来测量各种耗尽 NSUN m5C-MTase 的细胞中的 HIV 转录本，这将提供其 HIV 转录本的高分辨率分析此外，我们将确认 NSUN m5C- 的酶活性是否受到影响。我们还将确定 NSUN m5C-MTase 是否会干扰 HIV 治疗后。转录事件，包括 HIV mRNA 稳定性和翻译对于目标 2，我们将研究转录事件的影响。 NSUN m5C-MTase 激活 P-TEFb 和 RNA Pol-II，在促进 HIV 中发挥关键作用我们的结果表明，NSUN1 的缺失会降低 HIV TAR RNA 的 m5C 甲基化，并且其 MTase 催化结构域 (MTD) 可阻止 HIV Tat-TAR 相互作用，表明 TAR 的 m5C 甲基化可能直接调节其与 Tat 的相互作用我们将鉴定 TAR 催化的 m5C 甲基化位点。 NSUN m5C-MTases 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。我们将研究 NSUN m5C-MTase 在调节 HIV 5' 和 3' UTR 病毒功能中的作用。确定 NSUN m5C-MTase 是否与 HIV 5' 和 3' UTR 结合并对其 m5C 做出贡献由于 HIV mRNA 的 5' 和 3' UTR 在 HIV 前病毒表达调控中发挥着关键作用。在转录方面，我们将确定 HIV 5' 和 3' UTR 的 m5C 甲基化是否影响 mRNA 稳定性以及此外，最近发现 NSUN m5C-MTase 也发挥着重要作用。因此，我们将确定它们对宿主基因的功能影响。总体而言，该提案将全面研究 NSUN。 m5C-MTase 作为 HIV 原病毒表达的新型调节剂，我们相信这些研究将显着。提高我们对宿主与艾滋病毒相互作用的了解，我们将通过这些研究确认是否有成员。 NSUN m5C-MTase 可用于消除潜伏的 HIV。