Inactivation of infectivity of HIV-1 by compounds targeting nucleocapsid zinc finger motifs: mechanism(s) of action

The evolutionarily conserved zinc finger motifs in the nucleocapsid protein of retroviruses are essential for infectivity, with site directed mutagenesis studies implicating their involvement in RNA packaging, and in other as yet poorly understood, but critical preintegration processes. Chemical modification of the zinc fingers also inactivates infectivity, although the exact mechanisms involved have not been fully elucidated. We used the compound aldrithiol-2 to inactivate HIV-1 virus stocks, eliminating all detectable infectivity. Treatment of isolated HIV-1 NC p7 protein or virions followed by HPLC or immunoblot analysis gave results consistent with covalent modification of p7, with extensive crosslinking of p7 in treated virions. Studies were conducted to determine the stage of the viral life cycle at which the infectivity of the treated virions was arrested. Binding of treated virons to CD4+ target cells was comparable to binding for untreated native virions, however, using a quantitative real time PCR assay, we demonstrated that treated virons do not complete reverse transcription. This observation complements genetic studies in clarifying the functions of the NC protein. Studies to further define the exact stage of the viral life cycle at which infectivity is arrested for aldrithiol-2-treated virions will be presented, along with comparative studies using other compounds that target the p7 zinc fingers through different mechanisms. The conformational and functional integrity of virion surface proteins in aldrithiol-2 treated, non-infectious virions makes them an interesting candidate vaccine antigen, and provides additional incentive to understand the basis of their non-infectiousness.

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