Unique features of anthrax toxin are being exploited to make novel, cell-specific cytotoxins for HIV-1 infected cells. The three separate strategies described below for killing cells utilize highly toxic fusion proteins in which the amino-terminal portion of anthrax toxin lethal factor (LF) is genetically fused to the ADP-ribosylation domain of Pseudomonas exotoxin A (PE). Delivery of these LF-PE fusion proteins to the cytosol of cells requires the prior binding and proteolytic activation of the protective antigen (PA) component of the toxin. Three approaches to targeting cells are being used: 1. The site in PA which must be proteolytically cleaved is replaced by consensus sequences recognized by HIV-1 protease, to make a mutant PA that will be activated only in HIV-1-infected cells. Many of the mutant PA proteins produced were cleaved by HIV-1 protease in vitro. Surprisingly, several were toxic with LF for normal, non-infected cells, showing that endogenous proteases cleave within the newly added sequences. Additional target sequences will be constructed to find ones cleaved by the HIV-1 protease but not by endogenous cellular proteases. 2. CD4 and IL-2 are fused through a polypeptide linker to the carboxyl terminus of PA that has been altered to remove its ability to bind to its own receptor, either by truncation of the carboxyl terminus or mutagenesis of specific residues involved in receptor binding. 3. The gene encoding PA is transfected into several different mammalian cells to test the feasibility of sensitizing cells to LF fusion proteins by intracellular production of PA. The role of furin in processing HIV-1 gp160 is studied using hamster cells that are either normal, furin-deficient, or expressing furin from cDNA vector. Processing of gp160 is assessed biochemically and viral replication is tested by co-cultivation with virus-susceptible T cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Intramural Research (Z01)
Project #
1Z01DE000601-04
Application #
2572382
Study Section
Special Emphasis Panel (LME)
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
1996
Total Cost
Indirect Cost
Name
National Institute of Dental & Craniofacial Research
Department
Type
DUNS #
City
State
Country
United States
Zip Code