Long-term suppression of HIV-1C virus production in human peripheral blood mononuclear cells by LTR heterochromatization with a short double-stranded RNA
Long-term suppression of HIV-1C virus production in human peripheral blood mononuclear cells by LTR heterochromatization with a short double-stranded RNA
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DOI：
10.1093/jac/dkt348
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发表时间：
2014-02-01
影响因子：
5.2
通讯作者：
Chattopadhyay, Parthaprasad
中科院分区：
文献类型：
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作者：
Singh, Anand;Palanichamy, Jayanth K.;Chattopadhyay, Parthaprasad
整合的 HIV-1C 原病毒保守的 5 个长末端重复 (LTR) 启动子中的一个区域被鉴定为短双链 RNA (dsRNA) 的有效靶向，导致异染色质化，导致病毒转录、复制和随后在人类宿主细胞中的有效感染的长期减少。小干扰 RNA (siRNA) 被转染到含有整合的 siHa 细胞中 LTR-荧光素酶报告基因构建并筛选诱导转录基因沉默（TGS）的效率。通过双荧光素酶测定和实时 PCR 评估 TGS。还研究了目标区域的染色质修饰。然后检查有效 siRNA 在感染 HIV-1C 病毒的 TZM-bl 细胞和人外周血单核细胞 (PBMC) 中的有效性。通过 ELISA 测定病毒 Gag-p24 抗原水平。一种 HIV-1C LTR 特异性 siRNA 显着降低荧光素酶活性及其 mRNA 表达，但对 HIV-1B LTR 没有这种影响。这种 siRNA 介导的 TGS 是由组蛋白甲基化诱导的，从而导致目标 LTR 区域的异染色质化。相同的 siRNA 还可以在单次转染后 3 周内显着抑制感染各种 HIV-1C 临床分离株的 TZM-bl 细胞和人类 PBMC 中的病毒复制，甚至是目标区域不匹配的病毒株。我们已经鉴定出一种有效的 dsRNA，通过在目标 C-LTR 区域进行可遗传的表观遗传修饰，在体外和离体中长期抑制 HIV-1C 病毒的产生。这种 dsRNA 在 HIV-1C 感染中具有广阔的治疗潜力，全世界一半以上的艾滋病病例都是由 HIV-1C 感染引起的。
A region in the conserved 5 long terminal repeat (LTR) promoter of the integrated HIV-1C provirus was identified for effective targeting by a short double-stranded RNA (dsRNA) to cause heterochromatization leading to a long-lasting decrease in viral transcription, replication and subsequent productive infection in human host cells.Small interfering RNAs (siRNAs) were transfected into siHa cells containing integrated LTR-luciferase reporter constructs and screened for efficiency of inducing transcriptional gene silencing (TGS). TGS was assessed by a dual luciferase assay and real-time PCR. Chromatin modification at the targeted region was also studied. The efficacy of potent siRNA was then checked for effectiveness in TZM-bl cells and human peripheral blood mononuclear cells (PBMCs) infected with HIV-1C virus. Viral Gag-p24 antigen levels were determined by ELISA.One HIV-1C LTR-specific siRNA significantly decreased luciferase activity and its mRNA expression with no such effect on HIV-1B LTR. This siRNA-mediated TGS was induced by histone methylation, which leads to heterochromatization of the targeted LTR region. The same siRNA also substantially suppressed viral replication in TZM-bl cells and human PBMCs infected with various HIV-1C clinical isolates for 3 weeks after a single transfection, even of a strain that had a mismatch in the target region.We have identified a potent dsRNA that causes long-term suppression of HIV-1C virus production in vitro and ex vivo by heritable epigenetic modification at the targeted C-LTR region. This dsRNA has promising therapeutic potential in HIV-1C infection, the clade responsible for more than half of AIDS cases worldwide.