A major long term goal of this research is to develop an effective genetic therapy for AIDS. Specific plans include further testing of a retroviral vector encoding a molecule that blocks HIV-1 maturation. This molecule is a mutated soluble CD4 (sCD4-KDEL) that is retained in the endoplasmic reticulum (ER), binds the HIV-1 envelope protein, and prevents its transport to the cell surface. When placed under HIV-1 control in a retroviral vector, LHSN-CD4K, expression of the interfering protein is induced several hundred fold after HIV-1 infection, and assembly of infectious HIV-1 is completely blocked in a T cell line. The system is effective because of the high concentration of the blocking molecule (sCD4-KDEL) that accumulates in the ER. In addition, escape mutants may not be generated without losing the ability to bind receptor. Methods for transducing primary T lymphocytes with the LHSN-CD4K vector will be developed. After transduction, these cells can be challenged with primary isolates of HIV-1 to determine if their maturation is blocked also. These experiments are important because primary HIV-1 isolates have shown increased resistance to inhibition by soluble CD4. We will also determine if HIV-1 production can be halted by vector transduction into cells that are chronically infected by HIV-1. The mechanism of inhibition will be studied further by examining the non-infectious HIV-I particles produced to determine if they lack the envelope protein as anticipated. Additional studies will test the feasibility of generating defective particles that will specifically target HIV-infected cells and deliver the sCD4-KDEL gene. We will also examine the ability of the HIV-1 vpu protein to interfere with the action of sCD4-KDEL. Another goal is to determine if the retention in the ER of ternary complexes containing the HIV-1 envelope protein precursor, CD4, and a tyrosine kinase, plays a role in T cell killing.
