The objective of this proposal is to investigate the mutagenic effect of chronic treatment of human cells by anti-HIV therapeutic agents. A major emphasis of current medical research is the development of antiviral agents to prevent or reverse infection by human immunodeficiency virus (HIV), the etiologic agent of acquired immunodeficiency syndrome (AIDS). Studies of the HIV life cycle have targeted the reverse transcription of viral RNA to DNA as the most feasible stage for virus- specific therapeutic intervention. In the past three years, several deoxynucleoside analogues, used as inhibitors to the HIV reverse transcriptase, have been applied with some success to patients with AIDS and AIDS-related complex. The public pressure to expedite application of these components has, however, engendered concerns about their mutagenic effect on human host DNA replication and repair process during chronic treatment. These concerns prompt us to propose a study of the long term effects of these antiviral agents on DNA synthesis in human cells. Four specific experiments are proposed: 1. Development of human cell lines suitable for mutation studies. Following prolonged growth in media with antiviral compounds, cell clones will be screened for active nucleoside kinase activity and expanded into cell lines for the subsequent mutation studies. 2. Comparative analysis of mutations of selected human cells grown in control medium and in medium containing antiviral compounds by DNA synthesis forward mutation assays. 3. Molecular characterization of mutations. For those antiviral agents that cause high mutational frequencies, DNA synthesis fidelity of the principal replicative and repair DNA polymerase purified from cells chronically treated by these drugs will be analyzed. 4. Evaluation of the established and newly developed antiviral drugs by comparative inhibition studies of HIV reverse transcriptase and purified human DNA polymerase alpha, beta, and delta and analyze the DNA products of cellular polymerase with these inhibitors. The inhibition data correlated with the forward mutation analyses will provide invaluable reference for AIDS therapeutic strategy and biochemical rationale for the future antiviral therapeutic drug design.
| Copeland, W C; Lam, N K; Wang, T S (1993) Fidelity studies of the human DNA polymerase alpha. The most conserved region among alpha-like DNA polymerases is responsible for metal-induced infidelity in DNA synthesis. J Biol Chem 268:11041-9 |
| Chen, M S; Suttmann, R T; Papp, E et al. (1993) Selective action of 4'-azidothymidine triphosphate on reverse transcriptase of human immunodeficiency virus type 1 and human DNA polymerases alpha and beta. Biochemistry 32:6002-10 |
| Dong, Q; Copeland, W C; Wang, T S (1993) Mutational studies of human DNA polymerase alpha. Identification of residues critical for deoxynucleotide binding and misinsertion fidelity of DNA synthesis. J Biol Chem 268:24163-74 |
| Copeland, W C; Chen, M S; Wang, T S (1992) Human DNA polymerases alpha and beta are able to incorporate anti-HIV deoxynucleotides into DNA. J Biol Chem 267:21459-64 |
