Enzymatic analysis of two HIV-1 reverse transcriptase mutants with mutations in carboxyl-terminal amino acid residues conserved among retroviral ribonucleases H.
The reverse transcriptase (RT) of HIV-1 has been mutagenized within the carboxyl-terminal domain which harbors the RNase H. Two amino acids highly conserved among all 14 known RT sequences but not in the bacterial RNase H have been mutagenized resulting in the mutant proteins N494D and Q475E. They were expressed as recombinant proteins, purified, and analyzed for their in vitro properties in comparison to the p66 homodimeric wild-type and a previously described H539N mutant. The N494D mutant closely resembles the wild-type RNase H, exhibits an endonuclease activity and a processive RNase H activity, gives rise to small RNA hydrolysis products, and acts in concert with the RT. The Q475E mutant is more defective and resembles the H539N mutant, exhibits a retarded endonuclease activity and an impaired 3'-->5' processive RNA cleavage activity, gives rise to predominantly larger RNA hydrolysis products, is less processive in the presence of competitor substrate, and is defective in its ability to hydrolyze the polypurine tract and homopolymeric hybrids. Short homopolymeric stretches cause a pausing of the RT of wild-type and mutants which results in a coordinated action of the RNase H. Pausing of the RT correlates with RNase H cleavages about 20 nucleotides behind the point of synthesis. The defects of the mutant enzymes can be interpreted on the basis of the known crystallography data.
Volkmann
S
S
Max-Planck-Institut für Molekulare Genetik, Abteilung Schuster, Berlin, Germany.
Wöhrl
B M
BM
Tisdale
M
M
Moelling
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis; D054303:HIV Reverse Transcriptase; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013698:Templates, Genetic
D004247:DNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H