Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency.
Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency.
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DOI：
10.1186/s12977-022-00605-4
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发表时间：
2022-08-19
期刊：
影响因子：
3.3
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中科院分区：
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--
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通过选择性RNA剪接从一个初级转录本产生超过69个剪接的HIV-1mRNAs，强调了RNA处理在HIV-1复制中发挥的中心作用。控制在一定程度上是通过宿主SR蛋白的作用进行的，其活性受多个SR激酶(CLK1-4，SRPKs)的调节。在T细胞系和原代细胞中使用shRNA耗竭和宿主SR激酶的小分子抑制剂，以评估这些因素在HIV-1基因表达调控中的作用。用Western blotting、RT-qPCR和免疫荧光检测对病毒表达的影响。研究表明，SR激酶具有不同的作用；CLK1缺失促进HIV-1基因的表达，CLK2或SRPK1的缺失抑制其表达，而CLK3缺失对HIV-1基因表达的影响不大。CLK1和CLK2缺失的相反作用是由于不同步骤的作用；CLK1的减少增加了HIV-1启动子的活性，而CLK2的缺失影响了转录启动后的步骤。CLK1表达减少也增强了对几种潜伏期反转剂的反应，部分是通过增加反应细胞的频率，这与调节前病毒潜伏期的作用一致。为了确定小分子调节SR激酶功能是否可以用来控制HIV-1的复制，我们筛选了葛兰素史克蛋白激酶抑制剂文库，并鉴定了几种吡唑并[1，5-b]哒嗪衍生物，它们以EC50~ 50 nm抑制HIV-1基因的表达/复制。这些化合物抑制HIV-1蛋白和病毒RNA的积累，对细胞活力的影响最小，抑制CLK1和CLK2但不抑制CLK3的功能，从而选择性地改变细胞中单个CLK和SR蛋白的丰度。这些发现表明，单个SR激酶在调节HIV-1基因表达方面发挥着独特的作用，验证了这些功能的靶向性，以增强潜伏期逆转，这对于“踢和杀”策略是必不可少的，或为“阻止和锁定”策略沉默HIV蛋白的表达。网上版载有补充材料，可在10.1186/s12977-022-00605-4查阅。识别调节HIV-1RNA加工的细胞因素为控制这种感染的新策略提供了重要的见解。SR激酶家族的不同成员在调节病毒表达方面具有不同的作用，因为它们影响转录/RNA加工的不同步骤。我们在多个检测系统中发现了这些激酶的抑制剂，它们在纳摩尔浓度下抑制HIV-1基因的表达和复制，但细胞毒性有限或没有。我们的结果突出了针对HIV-1生命周期的整合后阶段的治疗潜力，以选择性地增强或逆转前病毒潜伏期。更好地了解观察到的影响背后的分子机制将有助于开发更有针对性的方法，以调节HIV-1潜伏期在通往这种感染的“功能性”治愈的道路上。网上版载有补充材料，可在10.1186/s12977-022-00605-4查阅。
The generation of over 69 spliced HIV-1 mRNAs from one primary transcript by alternative RNA splicing emphasizes the central role that RNA processing plays in HIV-1 replication. Control is mediated in part through the action of host SR proteins whose activity is regulated by multiple SR kinases (CLK1-4, SRPKs). Both shRNA depletion and small molecule inhibitors of host SR kinases were used in T cell lines and primary cells to evaluate the role of these factors in the regulation of HIV-1 gene expression. Effects on virus expression were assessed using western blotting, RT-qPCR, and immunofluorescence. The studies demonstrate that SR kinases play distinct roles; depletion of CLK1 enhanced HIV-1 gene expression, reduction of CLK2 or SRPK1 suppressed it, whereas CLK3 depletion had a modest impact. The opposing effects of CLK1 vs. CLK2 depletion were due to action at distinct steps; reduction of CLK1 increased HIV-1 promoter activity while depletion of CLK2 affected steps after transcript initiation. Reduced CLK1 expression also enhanced the response to several latency reversing agents, in part, by increasing the frequency of responding cells, consistent with a role in regulating provirus latency. To determine whether small molecule modulation of SR kinase function could be used to control HIV-1 replication, we screened a GSK library of protein kinase inhibitors (PKIS) and identified several pyrazolo[1,5-b] pyridazine derivatives that suppress HIV-1 gene expression/replication with an EC50 ~ 50 nM. The compounds suppressed HIV-1 protein and viral RNA accumulation with minimal impact on cell viability, inhibiting CLK1 and CLK2 but not CLK3 function, thereby selectively altering the abundance of individual CLK and SR proteins in cells. These findings demonstrate the unique roles played by individual SR kinases in regulating HIV-1 gene expression, validating the targeting of these functions to either enhance latency reversal, essential for “Kick-and-Kill” strategies, or to silence HIV protein expression for “Block-and-Lock” strategies. The online version contains supplementary material available at 10.1186/s12977-022-00605-4. Identifying cellular factors that regulate HIV-1 RNA processing provides important insights into novel strategies to control this infection. Different members of the SR kinase family have distinct roles in regulating virus expression because they affect distinct steps of transcription/RNA processing. We identify inhibitors of these kinases that suppress HIV-1 gene expression and replication in multiple assay systems at nanomolar concentrations with limited or no cytotoxicity. Our results highlight the therapeutic potential of targeting the post-integration stage of the HIV-1 lifecycle to selectively enhance or reverse provirus latency. A greater understanding of the molecular mechanisms underlying the effects observed will facilitate the development of more targeted approaches to modulate HIV-1 latency on the path toward a “functional” cure for this infection. The online version contains supplementary material available at 10.1186/s12977-022-00605-4.
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DOI：
10.1016/j.str.2008.12.023
发表时间：
2009-03-11
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
Bullock AN;Das S;Debreczeni JE;Rellos P;Fedorov O;Niesen FH;Guo K;Papagrigoriou E;Amos AL;Cho S;Turk BE;Ghosh G;Knapp S
通讯作者：
Knapp S
DOI：
10.3390/v14010060
发表时间：
2021-12-30
期刊：
Viruses
影响因子：
--
作者：
Dahal S;Cheng R;Cheung PK;Been T;Malty R;Geng M;Manianis S;Shkreta L;Jahanshahi S;Toutant J;Chan R;Park S;Brockman MA;Babu M;Mubareka S;Mossman K;Banerjee A;Gray-Owen S;Brown M;Houry WA;Chabot B;Grierson D;Cochrane A
通讯作者：
Cochrane A
影响因子：
3.3
作者：
Erkelenz S;Hillebrand F;Widera M;Theiss S;Fayyaz A;Degrandi D;Pfeffer K;Schaal H
通讯作者：
Schaal H
影响因子：
5.9
作者：
Cihlar, Tomas;Fordyce, Marshall
通讯作者：
Fordyce, Marshall
影响因子：
11.8
作者：
Guaraldi, Giovanni;Orlando, Gabriella;Palella, Frank
通讯作者：
Palella, Frank