Linker insertion mutagenesis of the human immunodeficiency virus reverse transcriptase expressed in bacteria: definition of the minimal polymerase domain.

• DOI: 10.1073/pnas.86.9.3104
• Publication Date: 2006-05-31T11:17:33Z
• Authors: V R Prasad; S P Goff
• Keywords: 570; 0303 health sciences; Molecular biology; Ribonuclease H; 610; HIV; RNA-Directed DNA Polymerase; DNA-Directed DNA Polymerase; 3. Good health; DNA polymerases; 03 medical and health sciences; Gene Expression Regulation; Virology; Endoribonucleases; Mutation; Escherichia coli; Transformation, Bacterial; Insertional mutagenesis; HIV (Viruses)--Treatment; Plasmids

A plasmid construct expressing the p66 version of the human immunodeficiency virus reverse transcriptase as a bacterial fusion protein was subjected to in vitro mutagenesis, and the resulting variant proteins were assayed to define the locations of the two major enzymatic activities. The DNA polymerase activity was localized to the N-terminal portion of the protein; mutations altering or eliminating the C-terminal portion had little or no effect on that activity. The results suggest that, in contrast with previous reports, the p51 subunit found in virions should exhibit DNA polymerase activity. Mutations in many parts of the protein eliminated RNase H activity, suggesting that several areas are needed for proper folding and generation of that activity.