INHIBITION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 ENV EXPRESSION BY C-5 PROPYNE OLIGONUCLEOTIDES SPECIFIC FOR REV-RESPONSE ELEMENT STEM-LOOP-V

The binding of Rev to the Rev-response element (RRE) of the human immu nodeficiency virus (HIV) is essential for RNA transport and expression of structural proteins such as gp160 encoded by env. To determine if env expression could be disrupted by complementary oligodeoxynucleotid es (ODNs), bandshift studies were used to identify RRE sites that are essential for the formation of Rev-RRE complexes [Chin, D. J. (1992) J . Virol. 66, 600-607] or the stability of preformed complexes. In this report, we describe complete disruption of preformed Rev-RRE complexe s by a subset of 15 ODNs complementary to stem-loop V. The most potent ODN complementary to bases CUGGGGCAUCAAGC disrupted 50% of preformed complexes at 1.2 mu M, a 400-fold molar excess over the RNA. Expressio n of env in COS7 cells was blocked by nuclear microinjection of ODNs w ith C-5 propyne-modified pyrimidines and phosphorothioate linkages. In hibition was highly dependent upon RNA target position, internucleotid e chemistry, ODN sequence, and concentration. Unmodified phosphodieste r or phosphorothioate ODNs were inactive. For the most potent ODN, 50% of the injected cells' env expression (I-50) was blocked with 0.1 mu M. A translational block is unlikely since these ODNs blocked expressi on of a luciferase vector in which the RRE was placed downstream of th e termination codon. Consistent with their in vitro effects upon Rev-R RE complexes, stem-loop V ODNs were 9-fold more active than stem-loop II ODNs in blocking env expression while having a reduced (I-50 = 0.27 mu M) but equivalent potency against luciferase-RRE. These results su ggest that disruption of Rev-RRE complexes may assist in blocking env expression.