Examining interactions of HIV-1 reverse transcriptase with single-stranded template nucleotides by nucleoside analog...

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• The p66 fingers subdomain of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) interacts with the template overhang.
• This interaction is crucial for accurate and efficient incorporation of deoxynucleoside triphosphates (dNTPs).

Purpose of the Study:

• To investigate the role of the template overhang's topology in DNA synthesis.
• To determine how specific mutations in the Phe-61 residue of HIV-1 RT affect DNA synthesis in response to template modifications.

Main Methods:

• Introduction of nucleoside analogs (e.g., abasic, methylphosphonate, conformationally locked) into the DNA template ahead of the catalytic center.
• Site-directed mutagenesis of the Phe-61 residue in HIV-1 RT (Phe-61 to Ala, Leu, Trp).
• Analysis of DNA synthesis pausing and bypass by the modified HIV-1 RT enzymes.

Main Results:

• Altering the template strand topology two nucleotides before the catalytic center interrupts DNA synthesis.
• Mutations of Phe-61 to Ala or Leu exacerbated the pausing effect caused by template analogs.
• Replacement of Phe-61 with tryptophan (Trp) enabled the mutant enzyme to bypass template analogs more readily.

Conclusions:

• The topology of the DNA template overhang significantly impacts HIV-1 RT processivity.
• The Phe-61 residue plays a critical role in mediating the enzyme's response to template structural alterations, influencing its ability to bypass or pause at modified template sites.