Son Jiyeon, Uchil Pradeep D, Kim Young Bong, Shankar Premlata, Kumar Priti, Lee Sang-Kyung
Department of Bioengineering, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791, Republic of Korea.
Biochem Biophys Res Commun. 2008 Sep 19;374(2):214-8. doi: 10.1016/j.bbrc.2008.06.125. Epub 2008 Jul 11.
The high genetic diversity and mutability of HIV pose a major problem for RNAi-mediated antiviral therapy. Simultaneous targeting of multiple highly conserved viral sequences has been suggested for durable cross-clade inhibition. Here we validate the approach of co-targeting two conserved sequences in the Tat and Vif genes. When coexpressed as artificial microRNA from a PolII driven miR-155-based vector, the sequences together mediated effective and sustained inhibition of HIV replication without virus breakout. To understand the nature of this efficient control, we analyzed genome sequences of 625 HIV-1 isolates in the Los Alamos Sequence database. Interestingly most natural variants were capable of wobble binding with the Tat/Vif siRNAs. Efficient silencing of reporter luciferase constructs bearing these variants residues verified that the Tat/Vif sequences together tolerated wobble binding and mediated functional RNAi. We propose the rationale of targeting highly conserved HIV sequences where wobble substitutions permit functional RNAi for global HIV repression.
HIV的高遗传多样性和变异性给RNA干扰介导的抗病毒治疗带来了重大问题。有人建议同时靶向多个高度保守的病毒序列以实现持久的跨亚型抑制。在此，我们验证了共靶向Tat和Vif基因中两个保守序列的方法。当从基于PolII驱动的miR-155载体作为人工微小RNA共表达时，这些序列共同介导了对HIV复制的有效且持续的抑制，且无病毒突破。为了解这种有效控制的本质，我们分析了洛斯阿拉莫斯序列数据库中625株HIV-1分离株的基因组序列。有趣的是，大多数天然变体能够与Tat/Vif小干扰RNA进行摆动碱基配对。对携带这些变体残基的报告荧光素酶构建体进行有效沉默，证实Tat/Vif序列共同耐受摆动碱基配对并介导功能性RNA干扰。我们提出了靶向高度保守的HIV序列的理论依据，其中摆动取代允许功能性RNA干扰以实现对全球HIV的抑制。
