Lee Sang-Kyung, Dykxhoorn Derek M, Kumar Priti, Ranjbar Shahin, Song Erwei, Maliszewski Laura E, François-Bongarçon Vanessa, Goldfeld Anne, Swamy N Manjunath, Lieberman Judy, Shankar Premlata
The CBR Institute for Biomedical Research, 800 Huntington Ave, Boston, MA 02115, USA.
Blood. 2005 Aug 1;106(3):818-26. doi: 10.1182/blood-2004-10-3959. Epub 2005 Apr 14.
Viral heterogeneity is a major hurdle for potential therapeutic use of RNA interference (RNAi) against HIV-1. To determine the extent of RNAi tolerance to mutations, we tested 3 viral target sites with differing propensity for mutations: a highly variable rev sequence, a gag sequence conserved only among clade B isolates, and a vif sequence highly conserved across clades. Lentiviral expression of all 3 shRNAs inhibited replication of the homologous HIV(IIIB) strain. However, they differed in their ability to protect primary CD4 T cells against multiple isolates within and across HIV clades. The least conserved rev sequence inhibited only 2 of 5 clade B isolates. The gag sequence (conserved within clade B) protected 5 of 5 clade B isolates but not other clade viruses with 2 or 3 mutations in the central region. In contrast, the vif sequence, which was conserved across clades except for single mutations at positions 14 and 17, inhibited viruses from 5 different clades. Moreover, siRNAs with introduced mutations at sites of gag sequence polymorphisms showed reduced antiviral activity, whereas mutations in vif siRNA only modestly decreased silencing. Thus, although 1 or 2 mutations at peripheral sites are tolerated, mutations in the central target cleavage region abolish RNAi activity.
病毒异质性是RNA干扰（RNAi）用于抗HIV-1治疗的一个主要障碍。为了确定RNAi对突变的耐受程度，我们测试了3个具有不同突变倾向的病毒靶点：一个高度可变的rev序列、一个仅在B亚型分离株中保守的gag序列以及一个在各亚型中高度保守的vif序列。所有3种短发夹RNA（shRNA）的慢病毒表达均抑制了同源HIV（IIIB）毒株的复制。然而，它们在保护原代CD4 T细胞免受HIV各亚型内及各亚型间多种分离株感染的能力上存在差异。保守性最低的rev序列仅抑制了5种B亚型分离株中的2种。gag序列（在B亚型内保守）保护了5种B亚型分离株中的5种，但对在中心区域有2或3个突变的其他亚型病毒则无保护作用。相比之下，vif序列除了在第14和17位有单个突变外，在各亚型中均保守，它抑制了来自5个不同亚型的病毒。此外，在gag序列多态性位点引入突变的小干扰RNA（siRNA）显示出抗病毒活性降低，而vif siRNA中的突变仅适度降低了沉默效果。因此，尽管周边位点的1或2个突变是可耐受的，但中心靶点切割区域的突变会消除RNAi活性。
