FAILURE TO SELECT FOR A DIBA RESISTANT MUTANT VIRUS IN ACUTELY AND CHRONICALLY HIV-1 INFECTED CELLS: IMPLICATIONS FOR ANTI-HIV-1 DRUG THERAPY

Abstract

The nucleocapsid (NCp7) protein of HIV-1 is required for multiple phases of the virus replication cycle. Characteristic of the NCp7 are two Cys-X₂-Cys-X₄-His-X₄-Cys arrays that bind zinc ions. These arrays are highly conserved throughout retroviral evolution. The high degree of conservation in the NCp7 creates an ideal target for the development of chemotherapeutic zinc finger antagonists. A recently discovered group of zinc finger antagonists are the disulfide-substituted benzamides (DIBAs). Their mechanism of action is to chemically attack the cysteine thiolate residues of the NCp7 zinc fingers. In turn, this electrophilic attack prompts zinc to be ejected from the zinc fingers. As a result of this alteration, the virus is non-infectious. After 4 months in culture, no DIBA resistant mutant viruses were selected by continuous passage in CEM-SS cells, syncytia sensitive clones derived from a human T4-Iymphoblastoid cell line. Continued cell passage for 18 months in the presence of HIV-1 and increasing concentrations of DIBAs did result in the creation of "DIBA-adapted" chronically infected CEM-SS cells. These cells continued to produce high levels of p24 and reverse transcriptase in the absence of syncytia formation. Electron microscopy revealed the presence of both mature and immature virus particles. Western blots of proteins derived from infected cells or virus particles displayed unprocessed, high molecular weight proteins in the Pr55ᵍᵃᵍ precursor polyprotein region. However, some processing was occurring as observed by the presence of p24 and NCp7 proteins. Experiments were performed to reduce the level of endogenous glutathione (GSH), thus promoting a more non-reducing environment. Such an environment might allow the DIBAs to more efficiently promote intracellular disulfide cross-linking among Gag precursors. Failure to generate a DIBA resistant mutant is a significant finding for antiretroviral therapy. In addition, further characterization of DIBAs in chronically infected cells becomes relevant for HIV-infected individuals.