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E.M. Cantin, P. Lundberg, J. Liao, M. Fan; A Role for RNAi in HSV–1 LAT Function . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3045.
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The available data suggest that LAT’s effects are mediated at the RNA level. Experiments were designed to test the hypothesis that LAT exerts its effects, which includes blocking apoptosis and possibly repression of lytic gene expression via encoded miRNAs.
Northern blot and RPA assays were used to detect small 21–25 nt RNAs in cells transfected with a LAT expression vector or infected with HSV–1. Potential miRNAs encoded in LAT were computationally predicted and possible targets were evaluated. The effect of silencing Dicer1 (Dcr1), an essential enzyme in RNAi, was used to determine whether LAT’s anti–apoptotic effects were mediated by miRNAs.
LAT–specific RNAs of 20–24 nt were highly expressed in a neuronal cell line transfected with a LAT expression vector or infected with HSV–1 but not a LAT deletion mutant. A potential LAT miRNA (miLAT) encoded in the region of overlap with ICP0 and present in HSV–2 also was predicted using "mirSeeker". Querying the miRNA registry with the putative miLAT showed that it is related to a cellular miRNA cloned from rat E18 cortical neurons. We identified a potential target site for miLAT that was conserved in ICP0 and a cellular Trim family gene. A synthetic LAT miRNA targeting this site silenced ICP0 resulting in reduced HSV–1 yields during low MOI infection. In transient transfection assays, the ability of the 2 kb LAT to inhibit apoptosis induced by treatment of Hela cells with Campothecin or anti–Fas antibody was blocked by a siRNA targeting Dcr1 but not an irrelevant siRNA.
These data suggest that LAT encodes at least two miRNAs and that it’s in vitro anti–apoptotic effects are Dcr1dependent and hence are mediated via the RNAi pathway.
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