Antibodies play a crucial role in humoral immunity. Physiologically, they are either expressed on the surface of B lymphocytes or secreted and, therefore act extracellularly. Recently, several sequence motifs responsible for intracellular localization of given proteins have been identified. As a result, it is becoming possible to target genetically engineered molecules into specific intracellular compartments. The object of this project is to determine whether antibodies can be directed into appropriate intracellular compartments and, if so, whether they will effect physiologic and/or pathologic processes. The model system that we are developing involves targeting antibodies to HIV gp41, p17, and protease into human CD4+T cells. HIV gp41 mediates fusion, oligomerization and viral budding processes. P17 is involved in virus budding and transporting the viral core from the cytosol to the nucleus. HIV protease is essential for uncoating and viral assembly. Thus, the multiple roles played by these proteins in the HIV life cycle suggest that they might be good targets for genetic intervention. We have made progress on several aspects of this project: 1) Anti-HIV-1 gp41 single chain Fv was targeted into the endoplasmic reticulum (ER) and transgolgi (TGN) of human CD4+T cells; 2) The targeted single chain Fv's were found to be stably expressed and bound to antigens; 3) These intracellular antibodies had significant inhibitory effect on the maturation of HIV-1 gp160 which, in turn, inhibited HIV-1 replication and syncytia. Recently, this project has been extended to a chemokine gene, interleukin-16 (IL-16). IL-16 is secreted from activated CD8+ T cells and acts upon CD4+T cells, monocytes, and eosinophils. The C-terminus of IL-16 was shown to suppress HIV-1 replication in vitro. To explore the potential of human IL-16 for gene therapy, stable CD4+ transfectants expressing human IL-16 were generated. The IL-16 in these transfectants inhibits HIV replication by as much as 99%. This represents the first demonstration of an HIV susceptible CD4+ cell line rendered resistant to HIV by transfection with a chemokine gene. In summary, we have developed an intracellular gene targeting approach to deliver heterologous proteins into the endoplasmic reticulum and transgolgi. The targeted antibodies and chemokine significantly inhibit HIV-1 replication. The application of this intracellular antibody gene approach is enormous. On one hand this approach could be used to determine the function of biologically important molecules, such as oncogenes, and on the other hand it could be used as a therapeutic tool to combat various diseases, such as cancer and HIV.
