Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation.

Abstract

Coupled with PCR, reverse transcriptases (RTs) have been widely used for RNA detection and gene expression analysis. Increased thermostability and nucleic acid binding affinity are desirable RT properties to improve yields and sensitivity of these applications. The effects of amino acid substitutions in the RT RNase H domain were tested in an engineered HIV-1 group O RT, containing mutations K358R/A359G/S360A and devoid of RNase H activity due to the presence of E478Q (O3MQ RT). Twenty mutant RTs with Lys or Arg at positions interacting with the template-primer (i.e., at positions 473–477, 499–502 and 505) were obtained and characterized. Most of them produced significant amounts of cDNA at 37, 50 and 65 °C, as determined in RT-PCR reactions. However, a big loss of activity was observed with mutants A477K/R, S499K/R, V502K/R and Y505K/R, particularly at 65 °C. Binding affinity experiments confirmed that residues 477, 502 and 505 were less tolerant to mutations. Amino acid substitutions Q500K and Q500R produced a slight increase of cDNA synthesis efficiency at 50 and 65 °C, without altering the KD for model DNA/DNA and RNA/DNA heteroduplexes. Interestingly, molecular dynamics simulations predicted that those mutations inactivate the RNase H activity by altering the geometry of the catalytic site. Proof of this unexpected effect was obtained after introducing Q500K or Q500R in the wild-type HIV-1BH10 RT and mutant K358R/A359G/S360A RT. Our results reveal a novel mechanism of RNase H inactivation that preserves RT DNA binding and polymerization efficiency without substituting RNase H active site residues.