Relationship between enzyme activity and dimeric structure of recombinant HIV-1 reverse transcriptase.

Area of Science:

• Biochemistry
• Molecular Biology
• Virology

Background:

• Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a crucial enzyme for viral replication.
• HIV-1 RT is a heterodimer comprising p66 and p51 subunits, possessing DNA polymerase and ribonuclease H (RNase H) activities.

Purpose of the Study:

• To investigate the structural and functional relationship between HIV-1 RT heterodimerization and its enzymatic activities.
• To elucidate the role of intersubunit interactions in the formation of the functional DNA polymerase active site.

Main Methods:

• Site-directed mutagenesis was used to introduce mutations at codon W401 of HIV-1 RT, inhibiting heterodimer formation.
• Enzymatic assays were performed to assess DNA polymerase and RNase H activities of wild-type (WT) and mutant enzymes.
• Cleavage patterns of RNase H activity were analyzed using various substrates.

Main Results:

• Mutations abrogating HIV-1 RT dimerization significantly inhibited DNA polymerase activity.
• p66 monomers showed only moderately reduced catalytic rates for both DNA polymerase-dependent and independent RNase H cleavage.
• No substantial alterations in RNase H cleavage patterns were observed between WT and mutant enzymes.

Conclusions:

• HIV-1 RT dimerization is essential for functional DNA polymerase activity.
• The p66 monomer can adopt an active conformation independent of dimerization.
• Intersubunit interactions within HIV-1 RT are critical for regulating the DNA polymerase active site.