Allosteric inhibition of the HIV-1 Rev/RRE interaction by a 3'-methylphosphonate modified antisense oligo-2'-O-methylribonucleotide.
The HIV-1 Rev response element (RRE), a highly structured RNA sequence consisting of five stemloops, is found in all spliced and partially spliced human immunodeficiency virus (HIV) mRNA transcripts. The RRE interacts with HIV-encoded Rev protein, which facilitates exit of the transcripts from the nucleus to the cytoplasm. Because the Rev/RRE interaction is critical to virus function, it is considered a potential target for therapeutic drugs. We have investigated the interactions of antisense oligonucleotides with stem-loop II, a region that contains the high-affinity binding site for Rev. Oligo-2'-O-methylribonucleotides terminating in a nuclease resistant 3'-methylphosphonate internucleotide linkage were targeted to the 5'- or 3'-side of stem-loop IIB, which is adjacent to the Rev binding site. Thermal denaturation experiments showed that oligonucleotides of this type form highly stable duplexes with complementary single-stranded RNA. Gel electrophoretic mobility shift assays (EMSA) showed that the oligonucleotides bound with high affinity and specificity at 37 degrees C to RRE stem-loop II RNA with apparent dissociation constants, K(D), in the low nM range. A 16-mer, 2-1mp, whose K(D) is 46 nM, competitively inhibited binding of Rev peptide to RRE stem-loop II RNA as shown by EMSA experiments. When transfected into HEK 293T cells, 2-1mp inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) by 60% at a concentration of 300 nM oligonucleotide. These results are consistent with a mechanism by which 2-1mp blocks access of Rev to the RRE/CAT transcript thus preventing nuclear export and subsequent translation.
Prater
Chrissy E
CE
Laboratory of Respiratory Biology, National Institutes of Environmental Sciences, Research Triangle Park, North Carolina, USA.
Saleh
Anthony D
AD
Wear
Maggie P
MP
Miller
Paul S
PS
D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D015694:Gene Products, rev / Q000378:metabolism*; D015752:Genes, env; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009690:Nucleic Acid Conformation; D020320:Oligoribonucleotides, Antisense / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*
D015694:Gene Products, rev; D020320:Oligoribonucleotides, Antisense; D012367:RNA, Viral