TY - JOUR
T1 - Allosteric inhibition of the HIV-1 Rev/RRE interaction by a 3′-methylphosphonate modified antisense oligo-2′-O- methylribonucleotide
AU - Prater, Chrissy E.
AU - Saleh, Anthony D.
AU - Wear, Maggie P.
AU - Miller, Paul S.
PY - 2007
Y1 - 2007
N2 - The HIV-1 Rev response element (RRE), a highly structured RNA sequence consisting of five stem-loops, 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°C to RRE stem-loop II RNA with apparent dissociation constants, KD, in the low nM range. A 16-mer, 2-1mp, whose KD 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.
AB - The HIV-1 Rev response element (RRE), a highly structured RNA sequence consisting of five stem-loops, 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°C to RRE stem-loop II RNA with apparent dissociation constants, KD, in the low nM range. A 16-mer, 2-1mp, whose KD 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.
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U2 - 10.1089/oli.2007.0082
DO - 10.1089/oli.2007.0082
M3 - Article
C2 - 17854268
AN - SCOPUS:34648830161
SN - 1545-4576
VL - 17
SP - 275
EP - 290
JO - Oligonucleotides
JF - Oligonucleotides
IS - 3
ER -