Role of RNA Methylation in Regulating HIV Proviral Expression

项目摘要 尽管HAART治疗成功地阻断了艾滋病患者体内HIV的活跃复制， 彻底根除感染艾滋病毒潜伏宿主仍然是彻底消除艾滋病毒的主要障碍。 艾滋病毒和治愈感染。调节HIV前病毒表达的宿主机制的研究 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， 扰乱宿主调节因子以消除潜伏的HIV。我们发现NSUN RNA m5 C 甲基转移酶（m5 C-MTases），NSUN 1/NOP 2，限制HIV复制，抑制HIV前病毒 表达，并促进病毒潜伏期。NSUN m5 C-MTases催化的m5 C甲基化的影响 （NSUN 1 -7）对HIV复制的影响仍然在很大程度上未知，但开始展开。在这份提案中，我们将发起 深入研究NSUN m5 C-MTases作为HIV前病毒表达的调节剂。对于目标1，我们将 采用超灵敏的逆转录液滴数字PCR（RT-ddPCR）测定来测量各种 NSUN m5 C-MTases耗尽的细胞中的HIV转录物，这将提供它们的高分辨率谱。 对HIV前病毒表达影响。此外，我们将确认NSUN m5 C-1的酶活性是否与NSUN m5 C-1的酶活性相关。 需要MTases。我们还将确定NSUN m5 C-MTases是否会干扰HIV感染后， 转录事件，包括HIV mRNA的稳定性和翻译。对于目标2，我们将研究 NSUN m5 C-MTases对P-TEFb和RNA Pol-II活化的影响，这些蛋白在促进HIV中发挥关键作用 转录。我们的研究结果表明，NSUN 1的缺失减少了HIV TAR RNA的m5 C甲基化， 其MTase催化结构域（MTD）阻止HIV Tat-TAR相互作用，表明TAR的m5 C甲基化 可能直接调节它与达特的相互作用。我们将鉴定由甲基化酶催化的TAR的m5 C甲基化位点。 NSUN m5 C-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 研究NSUN m5 C-MTases在调节HIV 5'和3' UTR '病毒功能中的作用。我们将 确定NSUN m5 C-MTases是否与HIV 5'和3' UTR结合以及是否有助于其m5 C 甲基化由于HIV mRNA的5'和3' UTR在HIV前病毒表达的调控中起关键作用， 在转录方面，我们将确定HIV 5'和3' UTR的m5 C甲基化是否影响mRNA的稳定性， 蛋白质翻译此外，最近已经显示NSUN m5 C-MTases也发挥重要作用。 在调节宿主细胞外转录组学中的作用。因此，我们将确定它们对宿主基因的功能影响 在HIV潜伏感染的CD 4 + T细胞中表达。总体而言，本提案将全面调查NSUN m5 C-MTases作为HIV前病毒表达的新型调节剂。我们相信，这些研究将大大 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，我们将确认 NSUN m5 C-MTases可用于消除潜伏的HIV。

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.