Funds are requested for a project aimed at defining critical nuclear HIV targets by a new injection assay that eliminates permeability assays. The investigator will attempt to test two conclusion that arise as a result of his preliminary studies. First, nuclear HIV sequences critical for replication may be blocked by oligonucleotides and, secondly, oligonucleotide efficacy to RNA targets may be improved by appropriate oligonucleotide chemistry. The applicant proposes to use both in vitro and in situ assays to establish the mechanism and specificity of antiviral effects. Sequences essential for HIV RNA processing and transport will be targeted with complimentary oligonucleotides. These include the RRE and CRS recently identified by the applicant. The primary goal of this application is to study the effects of individual anti-RRE oligonucleotides and optimal subregion sequences. Also to be studied are size and concentration effects and the role of oligonucleotides complementary to adjacent, """"""""non-essential"""""""" RRE sequences in destabilizing the RRE-rev interaction. These parameters will be studied by band shift studies, RNase H assays and RNase T1 protection studies. To avoid limitations of in vitro assays, the effects of A1 and other oligonucleotides on RRE function will be studied by in situ assays. The investigator proposes to disrupt the function of the RRE structure in situ by microinjecting oligonucleotides and monitoring reporter gene expression and RNA localization by either detecting injected immunogenic RNA in rev-expressing cells or in situ hybridization of env-expressing cells. Since the mechanism of action of anti-RRE oligonucleotides may be complex, the applicant also proposes to address the possibility that anti-RRE oligonucleotides facilitate RNA cleavage by endogenous RNase H activities. The abundance and structure of target RRE RNAs will be studied by primer-extension assays and amplification of the injected RNA transcripts by quantitative polymerase chain reaction (PCR). To cope with possible RNA degradation, turnover of injected RNA:oligonucleotide hybrids will be studied with co-injected RNasin in cells treated or untreated with Actinomycin D. However, a precedent for metastable endonucleolytic fragments of RNAs with strong secondary structure suggests that env RNA fragments will also be metastable.
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| Giachetti, C; Chin, D J (1996) Increased oligonucleotide permeability in keratinocytes of artificial skin correlates with differentiation and altered membrane function. J Invest Dermatol 107:256-62 |
| Fenster, S D; Wagner, R W; Froehler, B C et al. (1994) Inhibition of human immunodeficiency virus type-1 env expression by C-5 propyne oligonucleotides specific for Rev-response element stem-loop V. Biochemistry 33:8391-8 |
| Sixou, S; Szoka Jr, F C; Green, G A et al. (1994) Intracellular oligonucleotide hybridization detected by fluorescence resonance energy transfer (FRET). Nucleic Acids Res 22:662-8 |
