Title

Clinical and biochemical consequence of mutations in HIV-1 reverse transcriptase

Date of Award

1995

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Walter A. Scott, Committee Chair

Abstract

Prolonged therapeutic use of 3$\prime$-azido-2$\prime$, 3$\prime$-dideoxythymidine (AZT) in Acquired Immunodeficiency syndrome (AIDS) patients has allowed the development of strains of human immunodeficiency virus (HIV) resistant to inhibition by AZT (Larder et al. 1989). A number of mutations associated with the emergence of resistant virus have been mapped to the RT gene (Larder and Kemp, 1989, Larder et al. 1991). Mutations at codons 67, 70, 215, and 219 are persistently observed in patients undergoing AZT treatment. Introducing this set of four mutations to infectious viral clones results in 120-fold resistance to inhibition by AZT in cell culture (Kellam et al. 1992). Paradoxically, purified RT containing these mutations displays only 2-fold increase in the inhibition constant (K$\rm\sb{i}$) for AZTTP (Lacey et al. 1992).In the first section of our study we followed emergence of these mutations in 14 pediatric patients who were being treated with AZT. Correlations between the emergence of mutations and clinical and laboratory parameters were established. During the study period five patients developed mutation 70, four patients had 215, four patients had multiple mutations and one developed none. Statistical analysis of the clinical and laboratory data revealed that mutations 70 and 215 were associated with different clinical pictures. Children who were highly symptomatic developed mutation 215, and appearance of this mutation strongly correlated with poor clinical outcome. Children who were clinically stable developed mutation 70. We concluded that emergence of mutation 215 could be used as a predictive marker for imminent clinical deterioration, and these patients might benefit from a change in treatment regimen. Presence and persistence of mutation 70 might mean that conditions for developing mutation 215 are not met, and might be beneficial to the patient.The second part of our study focused on investigating biochemical differences between wild type RT and RT which contains mutations at codons 67 and 70. These mutations were introduced into the wild type RT gene through site-directed mutagenesis by PCR. Purified wild type (WRT) and mutant (MRT) RT's were used in primer extension assays on M13 DNA template to identify the differences between the enzymes. Our data suggests that MRT forms a more stable pre-synthesis complex with M13 primer-template than WRT. WRT and MRT showed different levels of discrimination between AZTTP and dTTP at different adenosines on the template. A model is proposed to explain AZTTP discrimination by MRT. We suggest that template-primer binding and the sequence of the template play important roles in the nucleotide selection process.

Keywords

Biology, Molecular; Chemistry, Biochemistry

Link to Full Text

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