Identification of the primer binding domain in human immunodeficiency virus reverse transcriptase

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Human Immunodeficiency Virus type 1 Reverse Transcriptase (HIV-1 RT) is essential for viral replication.
• Understanding the enzyme's primer binding domain is critical for developing antiviral therapies.
• Specific interactions between primers and HIV-1 RT dictate its enzymatic activity.

Purpose of the Study:

• To identify and characterize the primer binding domain of HIV-1 RT.
• To investigate the functional significance of primer cross-linking to the enzyme.
• To pinpoint the specific amino acid residues involved in primer binding and cross-linking.

Main Methods:

• UV-induced cross-linking of a 5'-32P-labeled (dT)15 primer to HIV-1 RT.
• Competition experiments using synthetic and natural primers to assess binding specificity.
• Enzyme activity assays to correlate primer binding with polymerase function.
• Peptide mapping using HPLC and DEAE-Sephadex chromatography to isolate cross-linked peptides.
• Amino acid composition and sequence analysis to identify cross-linked residues.

Main Results:

• Primer cross-linking to HIV-1 RT was specific and competed by other primers, including tRNA(Lys).
• tRNA(Lys) inhibited both polymerase activity and primer cross-linking to a similar extent, indicating functional relevance.
• Approximately 15% of the enzyme was cross-linked to the primer at a 1:3 enzyme to primer ratio.
• A single tryptic peptide containing residues 288-307 of HIV-1 RT was identified as the cross-linked domain.
• Leu(289)-Thr(290) and Leu(295)-Thr(296) were identified as probable sites of cross-linking.

Conclusions:

• The study successfully identified the primer binding domain of HIV-1 RT.
• Specific amino acid residues within the 288-307 region are critical for primer interaction.
• These findings provide valuable insights into the mechanism of HIV-1 RT and potential targets for drug development.