In vitro and in vivo characterization of primate lentiviral virions inactivated by targeting nucleocapsid protein zinc fingers

We used the disulfide reagent 2,2'-dithiodipyridine to inactivate the infectivity of HIV-1 and SIV virions by covalent modification of the essential zinc fingers in their nucleocapsid (NC) proteins. Western blot and HPLC analysis of treated virus preparations revealed extensive crosslinking of NC p7/p8 into high molecular weight aggregates. The inactivated virus was not detectably infectious in vitro (in excess of 5 logs inactivation). In contrast to matched virion preparations inactivated by conventional methods such as heat or formalin treatment, for virions inactivated by targeting NC zinc fingers both viral and host cell derived proteins on virion surfaces retained conformational and functional integrity, as assessed by immunoprecipitation, virion binding assays, and assays of virus mediated "fusion from without" and superantigen presentation by MHC Class II. However, quantitative PCR assays for reverse transcribed SIV DNA indicated that reverse transcription was not initiated by the treated virus following virion fusion with the target cell membrane. Inactivation via this method results in loss of infectivity with preservation of conformational and functional integrity of virion surface proteins. In preliminary studies, large amounts of inactivated SIV (equivalent to approximately 3 X 10(8) TCID50 units of native virus) have been infused in macaques (M. nemestrina, n=2); the inactivated virus appears to be non-infectious and immunogenic in vivo. Additional detailed studies of in vivo infectivity, immunogenicity, and protective vaccine efficacy are in progress. Current immunological and virological results will be presented.

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