PMID: 9435245Jan 22, 1998Paper

A genetic approach for identifying critical residues in the fingers and palm subdomains of HIV-1 reverse transcriptase

Proceedings of the National Academy of Sciences of the United States of America
J A WrobelC A Hutchison

Abstract

By using oligonucleotide-directed saturation mutagenesis, we collected 366 different single amino acid substitutions in a 109-aa segment (residues 95-203) in the fingers and palm subdomains of the HIV-1 reverse transcriptase (RT), the enzyme that replicates the viral genome. After expression in Escherichia coli, two phenotypic assays were performed. The first assay tested for RNA-dependent DNA polymerase activity. The other assay used Western blot analysis to estimate the stability of each mutant protein by measuring the processing of the RT into its mature heterodimeric form, consisting of a 66-kDa subunit and a 51-kDa subunit. The resulting phenotypic data provided a "genetic" means to identify amino acid side chains that are important for protein function or stability, as well as side chains located on the protein surface. Several HIV-1 RT crystal structures were used to evaluate the mutational analysis. Our genetic map correlates well with the crystal structures. Combining our phenotype data with crystallographic data allowed us to study the genetically defined critical residues. The important functional residues are found near the enzyme active site. Many residues important for the stability of the RT participate in potent...Continue Reading

References

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Citations

Jan 12, 2001·AIDS Research and Human Retroviruses·J A WrobelC A Hutchison
Mar 17, 2004·AIDS Research and Human Retroviruses·Maria G IsaguliantsBritta Wahren
Oct 9, 2007·Bioinformatics·Chris Deutsch, Bala Krishnamoorthy
May 26, 2009·Nucleic Acids Research·Antonin PavelkaJiri Damborsky
Jul 11, 2006·Annual Review of Genomics and Human Genetics·Pauline C Ng, Steven Henikoff
Jun 10, 2005·Genetics·Lev Y Yampolsky, Arlin Stoltzfus
Mar 29, 2000·Proceedings of the National Academy of Sciences of the United States of America·P L BoyerS H Hughes
Sep 10, 2015·Journal of Molecular Modeling·Kamil KhafizovSergei P Kovalenko
Apr 3, 1999·Viral Immunology·L Menéndez-AriasE Domingo
Mar 12, 2004·The Journal of Biological Chemistry·Robert A SmithBradley D Preston
Aug 31, 2004·The Journal of Biological Chemistry·Manjula PandeyAbram Gabriel
Nov 19, 2004·The Biochemical Journal·Clara E Cases-González, Luis Menéndez-Arias
Dec 15, 2020·Virology·Doty B A OjwachJaclyn K Mann

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