Mutational analysis of the DNA polymerase and ribonuclease H activities of human immunodeficiency virus type 2 reverse transcriptase expressed in Escherichia coli.
We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a polypeptide with an apparent molecular weight of 68 kDa. The HIV-2 reverse transcriptase (RT) made in E. coli is soluble in bacterial extracts and possesses both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activities typical of retroviral RTs. The HIV-2 RT expression clone was used to generate mutations in HIV-2 RT. There is a strong correlation between the effects of individual mutations on the DNA polymerase and RNase H activities. Mutations that profoundly affect the two catalytic functions are not clustered in any particular region of the polypeptide. Those few mutations that selectively affect either the RNase H or the DNA polymerase suggest that, like other retroviral RTs, the DNA polymerase is associated with the amino-terminal portion of HIV-2 RT and the RNase H with the carboxy-terminal portion. Genetically, the HIV-2 RT resembles the HIV-1 RT more closely than it resembles Moloney murine leukemia virus RT. The two catalytic functions of Moloney murine leukemia virus RT can be separately expressed in active form by molecular cloning; those of HIV-1 and HIV-2 RT cannot.
Hizi
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Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Israel.
Tal
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Hughes
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D000595:Amino Acid Sequence; D000818:Animals; D004252:DNA Mutational Analysis; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D015497:HIV-1 / Q000201:enzymology; D015498:HIV-2 / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010452:Peptide Biosynthesis; D010957:Plasmids / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D016914:Ribonuclease H; D012689:Sequence Homology, Nucleic Acid
D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H