Virion-targeted viral inactivation: New therapy against viral infection

Background: Acquired immune deficiency syndrome (AIDS) is resistant to all current therapy. Gene therapy is an attractive alternative or additive to c urrent, unsatisfactory AIDS therapy. Materials and Methods: To develop an antiviral molecule targeting viral int egrase (HIV IN), we generated a single-chain antibody, termed scAb, which i nteracted with human immunodeficiency virus type 1 (HIV-1) IN and inhibited virus replication at the integration step when expressed intracellularly. To reduce infectivity from within the virus particles, we made expression p lasmids (pC-scAbE-Vpr, pC-scAbE-CA, and pC-scAbE-WXXF), which expressed the anti-HIV IN scAb fused to, the N-terminus of HIV-1-associated accessory pr otein R (Vpr), capsid protein (CA), and specific binding motif to Vpr (WXXF ), respectively. All fusion proteins were tagged with a nine-amino acid pep titde derived from influenza virus hemagglutinin (HA) at the C terminus. Results: The fusion molecules, termed scAbE-Vpr, scAbE-CA, and scAbE-WXXF, interacted specifically with HIV IN immobilized on a nitrocellulose membran e. Immunoblot analysis showed that scAbE-Vpr, scAbE-CA, and scAbE-WXXF were incorporated into the virions produced by cotransfection of 293T cells wit h HIV-1 infectious clone DNA (pLAI) and pC-scAbE-Vpr, pC-scAbE-WXXF. A mult inuclear activation galactosidase indictor (MAGI) assay revealed that the v irions released fr om 293T cells cotransfected with pLAI and pC-scAbE-Vpr, pC-scAbE-WXXF had as little 1000-fold of the infectivity of the control wil d-type virions, which were produced from the 293T cells transfected with pL AI alone. Furthermore, the virions produced from the 293T cells cotransfect ed with pLAI and an scAb expression vector (pC-scAb) showed only 1% of the infectivity of the control HIV-1 in a MAGI assay, although scAb was not inc orporated into the virions. In either instance, the total quantity of the p rogeny virions released from the transfected 293T cells and the patterns of the virion proteins were hardly affected by the presence of scAb, scAbE-Vp r, or scAbE-WXXF, as determined by virion-associated reverse transcriptase assay and by immunoblot analysis, respectively. Because G418-selected HeLa clones carrying the expression plasmid for scAbE-WXXF were obtained much mo re frequently than those for scAbE-Vpr, scAbE-WXXF was inferred to be less toxic to cells than scAbE-Vpr. The result that scAbE-WXXF with viral incorp oration achieved more than a 10-fold reduction in infectivity of the progen y virions than scAb without incorporation suggests that scAbE-WXXF is a pot ential antiviral molecule, inhibiting replication by neutralization of: HIV IN activity both within cells and within virions. Moreover, it is nontoxic to human cells. We termed this gene therapy "virion-"targeted-viral inacti vation" and these molecules "packageable antiviral therapeutics." Conclusion: This new gene therapy has the potential for wide application in many viral infectious diseases.