The phosphorothioate oligonucleotide T2G4T2 was identflied as an inhibitor of HIV infection in vitro by combinatorial screening of a library of phosphorothioate oligonucleotides that contained all possible octanucleotide sequences. The oligonucleotide forms a parallel-stranded tetrameric guanosine-quartet structure. Tetramer formation and the phosphorothioate backbone are essential for antiviral activity. The tetramer binds to the human immunodeficiency virus envelope protein gpl20 at the V3 loop and inhibits both cell-to-cell and virus-to-cell infection. Since functional inhibition of viral infection rather than nucleic acid binding is the selection criterion, the chances are enhanced that a useful compound will be identified.When the SURF strategy was used in a functional screen for an inhibitor of HIV infection, phosphorothioate oligonucleotides with four consecutive guanosines were found to have activity. Of the active compounds, T2G4T2 was chosen for further studies. Physical characterization of this oligonucleotide demonstrated that it formed a tetramer stabilized by G quartets. G-quartet structures are intra-or intermolecular four-stranded helices stabilized by planar Hoogsteen-paired quartets of guanosine (8, 9). The structures are stabilized by monovalent ions bound between two planes ofG quartets and coordinated to the carbonyl oxygens. The G-quartet structure and a phosphorothioate backbone were shown to be required for antiviral activity.Cell culture experiments revealed that the G-quartet structure blocks binding of HIV virions to cells and virus-mediated cell fusion. Virus and cell membrane fusion is initiated by binding of gp120, the major envelope protein of HIV, to its cellular receptor. The mechanism by which membrane attachment occurs is not well understood, but a particular region of gp120, the V3 loop, is essential. The V3 loop maintains a high percentage ofpositively charged amino acids across all HIV strains (10), and the extent of virus-mediated cell fusion and rapid viral replication have been correlated with increased cationic composition of the V3 loop (11). In vitro studies showed that the G-quartet structure bound directly to the V3 loop of gpl20. We hypothesize that the G-quartet structure provides a scaffold to present the phosphorothioate groups in a favorable orientation for binding to the highly cationic V3 loop. MATERIALS AND METHODSOligonucleotide Synthesis. Phosphorothioate oligonucleotides were synthesized by standard protocols (12). For oligonucleotides with randomized positions, the proportions of the individual amidites in a mixture were adjusted until equal amounts of the four dimers were obtained when coupled to dT on controlled-pore glass, as judged by reversed-phase HPLC. Oligonucleotides were purified by reversed-phase HPLC with a gradient of methanol in water. Several purified oligonucleotides were analyzed for base composition by total digestion with nuclease followed by reversed-phase HPLC (Len Cummins, personal communication) and yielded the expected rati...

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The synthesis of 2',3' asymmetrically substituted pyrimidine ribonucleosides in 70-95% yields by using modified Verbruggen conditions with "nonparticipating" 2-0-CH3 and 2-O-TBDMS ribofuranoses is described. Such compounds are useful synthons for oligoribonucleotide synthesis, including incorporation of "rare" bases. New and practically useful conditions for placement (using 1,2,4-triazole) and removal (KF/crown ether) of the teri-butyldimethylsilyl (TBDMS) protecting group are also reported.

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