ABSTRACT
Reverse transcriptase, an essential retroviral DNA polymerase, replicates the single-stranded RNA genome of the retrovirus, producing a double-stranded DNA copy, which is subsequently integrated into the host's genome. Substitution of Ala for either Asp114 or Arg116, two highly conserved residues in the fingers domain of Moloney murine leukemia virus reverse transcriptase, results in enzymes (D114A or R116A) with significant defects in their abilities to processively synthesize DNA using RNA or DNA as a template. D114A and R116A enzymes also bind more weakly to template-primer in the presence of added deoxyribonucleotides, as seen by gel-shift analysis, but retain the ability to strand transfer and accumulate smaller RNase H cleavage products when compared to the wild-type enzyme. In addition, mutant proviruses, including D114A and R116A substitutions in Moloney murine leukemia virus reverse transcriptase, are not viable despite the presence of processed reverse transcriptase in the viral particles. A potential mechanistic role in processive synthesis for D114 and R116 is discussed in the context of our results, related studies on HIV-1 reverse transcriptase, and previous structural studies.
Subject(s)
Amino Acid Substitution/genetics , Arginine/metabolism , Aspartic Acid/metabolism , Moloney murine leukemia virus/enzymology , RNA-Directed DNA Polymerase/chemistry , RNA-Directed DNA Polymerase/metabolism , Arginine/genetics , Aspartic Acid/genetics , Base Sequence , Binding Sites , Blotting, Western , DNA/biosynthesis , DNA/chemistry , DNA/genetics , DNA/metabolism , HIV Reverse Transcriptase/metabolism , Models, Molecular , Molecular Sequence Data , Moloney murine leukemia virus/genetics , Moloney murine leukemia virus/physiology , Mutation/genetics , Nucleic Acid Conformation , Protein Binding , Protein Structure, Tertiary , Proviruses/enzymology , Proviruses/genetics , Proviruses/physiology , RNA/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Directed DNA Polymerase/genetics , Ribonuclease H/metabolism , Templates, Genetic , Viral Proteins/biosynthesis , Virion/enzymology , Virion/isolation & purification , Virion/physiology , Virus ReplicationABSTRACT
The case of a young female patient with pelvic intravenous leiomyomatosis extending into the right side of the heart successfully managed with tumor excision under cardiopulmonary bypass in staged procedure is presented.
