Title

Mutational analysis of the M41 residue of the human immunodeficiency virus-1 reverse transcriptase: Effects on reverse transcriptase activities and implications for drug resistance

Date of Award

2006

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Walter A. Scott, Committee Chair

Abstract

Human Immunodeficiency Virus-1 reverse transcriptase (HIV-1 RT) is an RNA-dependent, DNA-dependent polymerase, containing also an RNase H, and is responsible for the replication of the entire HIV-1 genome. RT replicates the genomic RNA into a double stranded DNA copy that is then integrated into the host genome.The kinetic reaction pathway for the DNA polymerization by HIV-1 RT has been established to be a sequential bireactant-biproduct mechanism. The rate-limiting step of the reaction is the conformational change associated with the binding of the next complementary dNTP right before the formation of the phosphodiester bond.HIV-1 RT is a target of drugs in the battle against AIDS (Acquired immunodeficiency syndrome) and the emergence of drug resistant variants has constituted a major hurdle in the control of the HIV infection. One such drug that does give rise to a host of resistant mutations in RT is 3'-azido-3'deoxythymidine (AZT). AZT is a nucleoside analog lacking a functional 3'-OH and acts as a chain terminator to block further DNA synthesis by HIV-1 RT. The basis of AZT resistance is the enhanced excision of the terminating AZTMP in the presence of an acceptor such as ATP. Resistance to AZT has been shown to be mediated by amino acid substitutions in the coding region of the HIV-1 RT and the T215Y mutation plays a central role in AZT resistance. The effect of this mutation in combination with the M41L mutation has been shown to cause >100 fold decrease in sensitivity to AZT, in vivo. In vitro studies have shown that while the M41L mutation by itself gives rise to a slight increase in resistance, the M41L/T215Y mutant RT shows a 9 fold increase in the removal activity of the enzyme with a ddAMP terminated template/primer and a 3 fold increase in the removal of an AZTMP from the primer terminus.This dissertation is an attempt to explain the mechanism underlying the synergism observed between the two residues M41 and T215 during the excision reaction and the effects of mutations at the 41 residue of HIV-1 RT on the activities of the enzyme. I have mutated the 41 codon using site-directed mutagenesis from a methionine to five different amino acids including leucine, isoleucine, arginine, tyrosine and aspartic acid. Viral isolates from patients have shown that while the most preferred substitution at the 41 position is with a leucine, changes to an isoleucine and an arginine also occur rarely. My results show that the mutation to a tyrosine results in an inactive enzyme. The mutation of this residue from a methionine to a leucine or an isoleucine result in removal activity effects similar to that observed with the wild type enzyme, but in the presence of the T215Y mutation, both double mutants led to an increase in the removal activity. However, I also detected a lowered DNA binding affinity with the M41I/T215Y mutation that suggests that this might be one of the reasons the leucine mutation is chosen over the isoleucine mutation at the residue M41 more frequently in nature. Studies with the arginine mutants suggest that the presence of the T215Y mutation can result in a decrease in the excision activity the enzyme. Thus, depending on the substitution at the residue 41, the T215Y mutation can lead to either an increase or decrease in the removal activities of RT.Processivity data also suggests that the effect of the T215Y mutation on this property of the enzyme is determined by the substitution at the M41 residue. The formation of stable ternary complexes with the mutant RTs showed that both the arginine and the aspartic acid substitutions had major effects. The aspartic acid mutants were found to be unable to form a ternary complex in the presence of foscarnet, a pyrophosphate analog. In the light of these results, we propose that effects on the removal activity are mediated via possible effects on the phosphate binding of the substrate for the removal reaction, such as ATP.

Keywords

Biology, Molecular; Chemistry, Biochemistry

Link to Full Text

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