AEGiS-15IAC: Trapping HIV-1 reverse transcriptase in the pretranslocation position by foscarnet (phosphonoformic acid, PFA).

15th International AIDS Conference

Bangkok, Thailand - July 11-16, 2004

Trapping HIV-1 reverse transcriptase in the pretranslocation position by foscarnet (phosphonoformic acid, PFA).

Int Conf AIDS 2004 Jul 11-16; 15:(abstract no. WeOrA1310)

Rutvisuttinunt W, Meyer PR, Matsuura SE, Rangarajan P, So AG, Scott WA
University of Miami School of Medicine, Miami, United States

BACKGROUND: HIV-1 reverse transcriptase (RT) has the ability to excise nucleoside RT inhibitors (NRTI) by a reaction that is related to pyrophosphorolysis. This excision reaction can only occur if RT is in an untranslocated position with the bond to be cleaved positioned in its active site and the primer terminus occupying the dNTP site. At this position, RT can transfer a nucleotide from the primer terminus to acceptors (e.g. PPi and nucleoside di- and triphosphates). This may provide a target for antiretroviral drugs that act by a mechanism distinct from NRTIs. Foscarnet (PFA), a PPi analog, is an example of this type of inhibitor.

METHODS: Formation of a stable complex with RT and a chain-terminated DNA primer-template was examined by electrophoretic mobility shift assay (EMSA). Positioning of RT relative to the primer terminus in these complexes was determined by DNase I and exonuclease protection assays using bacteriophage lambda and E. coli RecJ exonucleases.

RESULTS: RT forms a complex with PFA and a primer-template that is not disrupted by a heparin trap in the EMSA. The PFA complex could also be detected as discrete barriers to exonuclease digestion. The upstream and downstream barriers are located at position 30 and +6/+7 for the PFA complex and -29 and +7/+8 for the stable complex formed with the next complementary dNTP (+1 dNTP). Complex formation was confirmed by DNase I footprinting and the difference in positions between +1 and PFA complex was confirmed by a change in position of the predominant DNase I hypersensitive site in the footprint.

CONCLUSIONS: Interaction of HIV-1 RT binary complex with DNA primer-template is altered by interaction with either the +1 dNTP or PFA. PFA captures RT in a tight complex that is positioned one base pair upstream from its position in the complex with the +1 dNTP.

Keywords: AEGIS, HIV-1 Reverse Transcriptase, Foscarnet, HIV-1, DNA Primers, CD40 Ligand, Nucleosides, Nucleotides, Binding Sites, immunology

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