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 immunodeficiency 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 oligodeoxynucleotides (ODNs), band-shift 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 complexes 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 microM, a 400-fold molar excess over the RNA. Expression of env in COS7 cells was blocked by nuclear microinjection of ODNs with C-5 propyne-modified pyrimidines and phosphorothioate linkages. Inhibition was highly dependent upon RNA target position, internucleotide chemistry, ODN sequence, and concentration. Unmodified phosphodiester or phosphorothioate ODNs were inactive. For the most potent ODN, 50% of the injected cells' env expression (I50) was blocked with 0.1 microM. A translational block is unlikely since these ODNs blocked expression of a luciferase vector in which the RRE was placed downstream of the termination codon. Consistent with their in vitro effects upon Rev-RRE complexes, stem-loop V ODNs were 9-fold more active than stem-loop II ODNs in blocking env expression while having a reduced (I50 = 0.27 microM) but equivalent potency against luciferase-RRE. These results suggest that disruption of Rev-RRE complexes may assist in blocking env expression.
Fenster
S D
SD
Agouron Institute, La Jolla, California 92037.
Wagner
R W
RW
Froehler
B C
BC
Chin
D J
DJ
D000480:Alkynes / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D018076:DNA, Complementary / Q000494:pharmacology; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects*; D015686:Gene Products, env / Q000235:genetics / Q000378:metabolism; D018014:Gene Transfer Techniques; D015752:Genes, env; D015757:Genes, rev; D019302:HIV Envelope Protein gp160; D015497:HIV-1 / Q000235:genetics*; D008845:Microinjections; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics
D000480:Alkynes; D018076:DNA, Complementary; D004279:DNA, Viral; D015686:Gene Products, env; D019302:HIV Envelope Protein gp160; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; C022030:methylacetylene; D016914:Ribonuclease H; D001616:beta-Galactosidase