Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
PLoS Pathog. 2020 Feb 18;16(2):e1008307. doi: 10.1371/journal.ppat.1008307. eCollection 2020 Feb.
The ability of HIV-1 to evolve resistance to combined antiretroviral therapies (cARTs) has stimulated research into alternative means of controlling this infection. We assayed >60 modulators of RNA alternative splicing (AS) to identify new inhibitors of HIV-1 RNA processing-a segment of the viral lifecycle not targeted by current drugs-and discovered compound N-[4-chloro-3-(trifluoromethyl)phenyl]-7-nitro-2,1,3-benzoxadiazol-4-amine (5342191) as a potent inhibitor of both wild-type (Ba-L, NL4-3, LAI, IIIB, and N54) and drug-resistant strains of HIV-1 (IC50: 700 nM) with no significant effect on cell viability at doses tested. 5342191 blocks expression of four essential HIV-1 structural and regulatory proteins (Gag, Env, Tat, and Rev) without affecting total protein synthesis of the cell. This response is associated with altered unspliced (US) and singly-spliced (SS) HIV-1 RNA accumulation (60% reduction) and transport to the cytoplasm (loss of Rev) whereas parallel analysis of cellular RNAs revealed less than a 0.7% of host alternative splicing (AS) events (0.25-0.67% by ≥ 10-20%), gene expression (0.01-0.46% by ≥ 2-5 fold), and protein abundance (0.02-0.34% by ≥ 1.5-2 fold) being affected. Decreased expression of Tat, but not Gag/Env, upon 5342191 treatment was reversed by a proteasome inhibitor, suggesting that this compound alters the synthesis/degradation of this key viral factor. Consistent with an affect on HIV-1 RNA processing, 5342191 treatment of cells altered the abundance and phosphorylation of serine/arginine-rich splicing factor (SRSF) 1, 3, and 4. Despite the activation of several intracellular signaling pathways by 5342191 (Ras, MEK1/2-ERK1/2, and JNK1/2/3), inhibition of HIV-1 gene expression by this compound could be reversed by pre-treatment with either a G-protein α-subunit inhibitor or two different MEK1/2 inhibitors. These observations demonstrate enhanced sensitivity of HIV-1 gene expression to small changes in host RNA processing and highlights the potential of modulating host intracellular signaling as an alternative approach for controlling HIV-1 infection.
HIV-1 能够进化出对联合抗逆转录病毒疗法（cART）的耐药性，这激发了人们对控制这种感染的替代方法的研究。我们检测了 >60 种 RNA 可变剪接（AS）调节剂，以鉴定新的 HIV-1 RNA 加工抑制剂-这是病毒生命周期中当前药物未针对的一个环节-并发现化合物 N-[4-氯-3-(三氟甲基)苯基]-7-硝基-2,1,3-苯并恶二唑-4-胺（5342191）是一种有效的野生型（Ba-L、NL4-3、LAI、IIIB 和 N54）和耐药性 HIV-1 株的抑制剂（IC50：700 nM），在测试剂量下对细胞活力没有显著影响。5342191 阻断了四种必需的 HIV-1 结构和调节蛋白（Gag、Env、Tat 和 Rev）的表达，而不影响细胞的总蛋白合成。这种反应与未剪接（US）和单剪接（SS）HIV-1 RNA 积累的改变有关（60%减少）和细胞质转运（Rev 丢失），而对细胞 RNA 的平行分析显示，宿主可变剪接（AS）事件的变化小于 0.7%（0.25-0.67%，≥10-20%），基因表达的变化小于 0.46%（0.01-0.46%，≥2-5 倍），蛋白质丰度的变化小于 0.34%（~0.02-0.34%，≥1.5-2 倍）。在用 5342191 处理后，Tat 的表达减少，但 Gag/Env 没有减少，这一现象被蛋白酶体抑制剂所逆转，表明这种化合物改变了这种关键病毒因子的合成/降解。与 HIV-1 RNA 加工的影响一致，5342191 处理细胞改变了丝氨酸/精氨酸丰富的剪接因子（SRSF）1、3 和 4 的丰度和磷酸化。尽管 5342191 激活了几种细胞内信号通路（Ras、MEK1/2-ERK1/2 和 JNK1/2/3），但这种化合物对 HIV-1 基因表达的抑制作用可以被 G 蛋白α亚单位抑制剂或两种不同的 MEK1/2 抑制剂的预处理所逆转。这些观察结果表明，HIV-1 基因表达对宿主 RNA 加工的微小变化更加敏感，并突出了调节宿主细胞内信号作为控制 HIV-1 感染的替代方法的潜力。
