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Mutations in the primer grip of human immunodeficiency virus type 1 reverse transcriptase impair proviral DNA
1LaboRetro, Unité de Virologie Humaine INSERM U412, Ecole Normale Supérieure de Lyon, 69364 Lyon Cedex 07, France.
Abstract:
This report describes the effects of mutating highly conserved residues in the primer grip domain of human immunodeficiency virus type 1 reverse transcriptase (RT) on virus formation and infectivity. Among a series of RT mutant viruses, three (M230A, L234D, and W239A) were found to be noninfectious or very poorly infectious. Our data indicate that these mutations in RT caused severe defects in proviral DNA synthesis. Interestingly, assembly and maturation of mutant virus M230A were similar to those of the wild type, while mutants L234D and W239A showed impaired maturation. The immature morphology of RT mutants L234D and W239A is due at least in part to premature cleavage of the gag-pol precursor, prior to virion budding, indicating that intracellular stability of Pr160(gag-pol) is of key importance during virus assembly.
Insights
Mutations in human immunodeficiency virus type 1 reverse transcriptase (RT) primer grip domain severely impair viral DNA synthesis and infectivity. Some mutations also disrupt gag-pol precursor cleavage, affecting virus maturation and assembly.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- The primer grip domain of human immunodeficiency virus type 1 reverse transcriptase (RT) is crucial for viral replication.
- Understanding the role of conserved residues in this domain is key to developing antiviral strategies.
Purpose of the Study:
- To investigate the impact of mutating highly conserved residues in the RT primer grip domain on human immunodeficiency virus type 1 (HIV-1) formation and infectivity.
- To elucidate the specific defects caused by these mutations in viral DNA synthesis, assembly, and maturation.
Main Methods:
- Site-directed mutagenesis was used to introduce mutations in conserved residues of the HIV-1 RT primer grip domain.
- Analysis of viral infectivity, proviral DNA synthesis, and virion morphology was performed for mutant viruses.
- Western blotting was employed to assess gag-pol precursor cleavage and intracellular stability.
Main Results:
- Three specific mutations (M230A, L234D, W239A) resulted in noninfectious or poorly infectious viruses.
- These mutations caused severe defects in proviral DNA synthesis.
- While M230A showed normal assembly and maturation, L234D and W239A exhibited impaired maturation due to premature gag-pol precursor cleavage.
Conclusions:
- Highly conserved residues in the HIV-1 RT primer grip domain are essential for viral infectivity and DNA synthesis.
- Intracellular stability of the Pr160(gag-pol) precursor is critical for proper virus assembly and maturation.
- Premature gag-pol cleavage contributes to immature virion morphology and reduced infectivity.