In vivo studies of in vitro selected HIV-1 Rev aptamers (Immune deficiency)

Human immunodeficiency virus type 1 (HIV-l) expresses a regulatory protein, Rev, which plays an essential role in the viral life cycle. Rev facilitates the nuclear export of incompletely spliced viral mRNAs resulting in the production of viral structural proteins. Therefore Rev is required for the production of progeny virions. Rev interacts with the Rev responsive element (RRE) on the viral mRNA. Within the RRE, the primary Rev binding site is the Rev binding element(RBE). The Rev:RBE interaction has been studied by in vitro selection. Aptamers (RNA decoys) with higher than wild-type affinities for Rev were selected from randomized RNA populations. Whether the high-affinity aptamers would bind Rev in vivo was unknown. The goal of this research was to study the in vivo Rev-binding and Rev-inhibiting activity of in vitro selected RNA aptamers in tissue culture systems.; A Rev-dependent reporter assay was utilized to address the in vivo activities of the in vitro selected aptamers. Cells were co-transfected with a Rev expression plasmid and a reporter plasmid (pDM 128) containing an intron with the RRE adjacent to a reporter gene. Upon binding to the RRE, Rev inhibits splicing and facilitates nuclear export of the RNA, resulting in detectable reporter activity. Aptamers were substituted into the RRE of pDM128, replacing the wild-type RBE. These hybrid RREs were expressed in cells and shown to support Rev function. When Rev was limiting, the hybrid RREs were more Rev-responsive than the wild-type RRE. This was the first demonstration of artificially evolved sequences functionally substituting for a wild-type element in vivo. The assay was similarly used to test whether RBEs in tandem can functionally substitute for the RRE. Additional RBEs supported RNA transport leading to graded Rev response.; Aptamer sequences may serve as therapeutic decoys by sequestering Rev and inhibiting the Rev:RRE interaction. Cellular Rev inhibition assays standardized to RNA expression levels revealed that the efficacy of aptamer sequences transcends that of the RBE. This research begins defining the applicability of in vitro selected aptamers for the in vivo disruption of viral protein:RNA interactions.