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Interaction of tRNA(Lys-3) with multiple forms of human immunodeficiency virus reverse transcriptase
N J Richter-Cook1, K J Howard, N M Cirino
1Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
The Journal of Biological Chemistry
|August 5, 1992
Summary
Dimerization of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is essential for efficient primer binding. Monomeric forms (p66 and p51) show weak binding, but heterodimer formation restores activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Virology
Background:
- Recombinant human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) exists in several forms, including p51, p66, and the heterodimer p66/p51.
- HIV-1 RT plays a crucial role in viral replication by synthesizing viral DNA from its RNA genome.
- Understanding the interaction between HIV-1 RT and its replication primer is vital for developing antiviral therapies.
Purpose of the Study:
- To investigate the binding affinity of different forms of recombinant HIV-1 RT (p51, p66, p66/p51) to its cognate primer tRNA(Lys-3).
- To determine the role of dimerization in the enzymatic activities of HIV-1 RT, specifically tRNA binding and RNA-dependent DNA polymerase activity.
Main Methods:
- Gel-mobility shift analysis was employed to study the interaction between HIV-1 RT forms and tRNA(Lys-3).
- Gel permeation analysis was used to assess the oligomeric state of purified p66 RT.
- Incubation of monomeric subunits was performed to observe heterodimer reconstitution and functional recovery.
Main Results:
- The heterodimeric form (p66/p51) of HIV-1 RT demonstrated proficient tRNA binding.
- Monomeric forms (p66 and p51) exhibited weak tRNA binding, even at high protein concentrations.
- Gel permeation indicated that purified p66 RT is predominantly monomeric, suggesting dimerization is necessary for efficient tRNA binding.
- Heterodimer reconstitution upon incubation restored both tRNA binding and RNA-dependent DNA polymerase activity.
- Monomeric p66 and p51 polypeptides retained their respective activities and did not bind tRNA efficiently under the same incubation conditions.
Conclusions:
- Dimerization of HIV-1 RT is a prerequisite for efficient binding to its replication primer, tRNA(Lys-3).
- The enzymatic activities, including RNA-dependent DNA polymerase and tRNA binding, are significantly enhanced in the heterodimeric form compared to monomeric forms.
- These findings highlight the importance of the p66/p51 heterodimer structure for the functional integrity of HIV-1 RT in viral replication.