کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2185059 1095958 2011 13 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
RNA Interference as a Tool for Exploring HIV-1 Robustness
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیولوژی سلول
پیش نمایش صفحه اول مقاله
RNA Interference as a Tool for Exploring HIV-1 Robustness
چکیده انگلیسی

Short interfering RNAs (siRNAs) that target viral genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, there is the potential for viral escape, particularly with a highly mutable target such as HIV-1. We present a novel strategy for anticipating and preventing viral escape using second-generation siRNAs. The evolutionary capacity of HIV-1 was tested by exerting strong selective pressure on a highly conserved sequence in the HIV-1 genome. We assayed the antiviral efficacy of five overlapping siRNAs directed against an essential region of the HIV-1 protease. Serial viral transfers in U87-CD4-CXCR4 cells were performed using four of the siRNAs. This procedure was repeated until virus breakthrough was detected. After several serial culture passages, resistant virus with a single point mutation in the targeted region was detected in the culture supernatants. The emergence of resistant virus was confirmed by molecular cloning and DNA sequencing of viral RNA. The most common escape route was the D30N mutation. Importantly, the addition of a second-generation siRNA that matched the D30N mutation restored viral inhibition and delayed development of escape variants. Passages performed with both siRNAs prevented the emergence of the D30N escape mutant and forced the virus to develop new escape routes. Thus, second-generation siRNAs can be used to block escape from RNA interference (RNAi) and to search for new RNAi escape routes. The protocol described here may be useful for exploring the sequence space available for HIV-1 evolution and for producing attenuated or deleterious viruses.


► We present a novel strategy for anticipating and preventing viral escape using second-generation siRNAs.
► Second-generation siRNAs block escape from RNAi and force the virus to develop new escape routes.
► Second-generation siRNAs allow the exploration of the sequence space available for virus evolution and for producing attenuated or deleterious viruses.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Molecular Biology - Volume 413, Issue 1, 14 October 2011, Pages 84–96
نویسندگان
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