Stepwise mechanism of HIV reverse transcriptase: primer function of phosphorothioate oligodeoxynucleotide

Abstract

Primer recognition by purified HIV reverse transcriptase has been investigated. Earlier we found that the reaction pathway for DNA synthesis is ordered, with template-primer and free enzyme combining to form the first complex in the reaction sequence (Majumdar et al., 1988). We now find that d(C)28 is a linear competitive inhibitor of DNA synthesis against poly[r(A)].oligo[d(T)] as template.primer, indicating that d(C)28 and the template.primer combine with the same form of the enzyme in the reaction scheme, i.e., the free enzyme. The phosphorothioate oligodeoxynucleotide Sd(C)28 also is a linear competitive inhibitor against template.primer. However, the Ki for inhibition (approximately 2.8 nM) is approximately 200-fold lower than the Ki for inhibition by d(C)28. Since the inhibition is linear competitive, the dissociation constant is equal to the Ki for inhibition. Filter binding assays confirmed high-affinity binding between Sd(C)28 and the enzyme and yielded a KD similar to the Ki for inhibition. Substrate kinetic studies of DNA synthesis using Sd(C)28 as primer, and poly[r(I)] as template, revealed that the Km for Sd(C)28 is 24 nM. The Km for this primer is, therefore, 8-fold higher than the KD for enzyme-primer binding (2.8 nM). These results enable calculation of real time rate values for the enzyme-primer association (kon = 5.7 x 10(8) M-1 s-1) and dissociation (koff = 1.6 s-1).

High‐Affinity Interaction of Human Immunodeficiency Virus Type‐1 Reverse Transcriptase with Partially Complementary Primers

“…According to Majumdar et al (1988Majumdar et al ( , 1989) and our results (Andreola et al, 1993), HIV-1 RT is able to bind the primer before the template. We have shown that, in the absence of template, HIV-1 RT could correctly recognize the 3'9erminal nucleotide of primers bearing chemically reactive groups at the 5' end.…”

Antisense oligonucleotides in solution or encapsulated in immunoliposomes inhibit replication of HIV-1 by several different mechanisms

“…No targeted or untargeted liposome-encapsulated oligonucleotides inhibited syncytium formation (Fig. 1) when tested at a final concentration of 0.5 A.M, which is the concentration at which they efficiently inhibit HIV-1 replication (25 (19,20). The total incubation time post-infection was 7 days.…”

“…Phosphorothioate oligonucleotides have been shown to inhibit the replication of HIV-1 in vitro by both sequence-non-specific (3,5,10,11) and specific processes (12)(13)(14)(15)(16), depending on the type of infection. In acutely infected cells, phosphorothioate oligonucleotides acted in a sequence-non-specific fashion probably by inhibition of viral binding and fusion (17,18) and/or viral reverse transcriptase (RT) (19,20). In chronically infected cells, however, only sequencespecific oligonucleotides are active, since after integration viruscell binding and reverse transcription are no longer relevant (21).…”

“…In contrast, inhibition by oligonucleotides of HIV-1 reverse transcriptase-dependent DNA synthesis has been well studied in cell-free systems. Phosphorothioate oligonucleotides are competitive inhibitors of primer binding in a manner largely independent of their nucleotide sequences (19,20). Phosphodiester oligonucleotides inhibit reverse transcription in a sequence-specific manner, indicating that their activity depends on interaction with the viral genome (27,28).…”

Specific inhibition of human telomerase activity by transfection reagent,FuGENE6‐antisense phosphorothioate oligonucleotide complex inHeLa cells

“…Presumably, addition bases of the longer S-ODNs offer more potential for non-specific contacts in the yield stronger intramolecular secondary structure, thereby reducing the likelihood of binding the telomerase target. It is known that the phosphorothioate oligonucleotides blocked the proliferation HIV-1 in infected cells in a nonsequence specific manner [43], probably the inhibition of reverse transcriptase [44,45] and/or the viral entry process [46,47]. Our previous study of the anti-HIV activities of antisense oligonucleotides indicated that a phosphorothioate oligonucleotide targeted to the HIV-1 gag gene was inhibitory, and the sense and the random sequence oligomers were also able to protect against HIV-1 induced CPE, but their anti-HIV-1 activities were weaker than that of the antisense phosphorothioate oligonucleotide (S-ODN-gag-AUG) [48].…”