Primary, symptomatic HIV-1 infection is associated with high titers of cytopathic, replication-competent viral strains, and during such infection potential infectivity is enhanced. Effective control of HIV-1 replication during primary infection implies the activation of clinically important mechanisms of immune defense that merit further examination in relation to the development of antiviral therapy and vaccines.

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...

No abstract

We have previously reported identification of a phosphorothioate oligonucleotide TTGGGGTT (ISIS 5320) as a potent inhibitor of HIV infection in vitro. The oligonucleotide forms a parallel-stranded, tetrameric guanosine quartet (G-quartet) structure that specifically binds to the HIV envelope glycoprotein (gp120) and inhibits both cell-to-cell and virus-to-cell infection at submicromolar concentrations. In the current study we demonstrate that the tetramer inhibits the infection of laboratory-derived isolates of HIV-1 and HIV-2 in a variety of phenotypically distinct, established human cell lines and a panel of biologically diverse clinical isolates in fresh human peripheral blood lymphocytes and macrophages. The compound was also active against all drug-resistant virus isolates tested. In combination with AZT, ISIS 5320 exhibits additive to slightly synergistic anti-HIV activity. Cell-based mechanism of action studies demonstrate that the compound inhibits the binding of infectious virus and virus-infected cells to uninfected target cells by binding to the cationic V3 loop of the envelope glycoprotein. The G-quartet structure is a potential candidate for use in anti-HIV chemotherapy.

No abstract