Source
Laboratory of Experimental Virology, Department of Medical Microbiology and Centre for Infection and Immunity Amsterdam (CINIMA), Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
Abstract
BACKGROUND:
RNA interference (RNAi) is a cellular mechanism that can be induced by small interfering RNAs to mediate sequence-specific gene silencing by cleavage of the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short hairpin RNAs (shRNAs). Previously, we used a co-transfection assay in which shRNA constructs were transfected with an HIV-1 molecular clone to identify 20 shRNA inhibitors that target highly conserved HIV-1 sequences.
METHODS:
In the present study, we selected the most potent shRNAs to formulate a combinatorial shRNA therapy and determine the best and easiest method for antiviral shRNA selection. We performed transient inhibition assays with either a luciferase reporter or HIV-1 molecular clone and also infected shRNA-expressing T cell lines with HIV-1 and monitored virus replication. The latter assay allows detection of viral escape. In addition, we also tested shRNA-expressing T cells upon challenge with increasing dosages of HIV-1, and measured the dose required to result in massive virus-induced syncytia formation in this 2-week assay.
RESULTS:
Extended culturing selected three highly effective shRNAs that do not allow viral replication for more than 100 days. This difference in potency was not observed in the transient co-transfection assays. The use of increased dosages of HIV-1 selected the same highly potent shRNAs as the laborious and extended escape study.
CONCLUSIONS:
These highly potent shRNAs could be used for a clinical vector and the comparison of the developed assays might help other researchers in their search for antiviral shRNAs.
(c) 2009 John Wiley & Sons, Ltd.