Mutational analysis of the fingers and palm subdomains of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase.
We have analyzed the human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) polymerase domain between amino acids 91 and 157 by site-directed mutagenesis. We have constructed a series of amino acid substitutions using BspMI cassettes, and have assayed the RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities of the mutant HIV-1 RTs. The regions of HIV-1 RT between amino acids 91 and 119 and between amino acids 151 and 157 lie within the palm subdomain and include part of the polymerase active site. A number of amino acids within these regions have been identified as being directly or indirectly involved with polymerization, since amino acid substitutions at these residues decrease the polymerase activity without affecting RNase H activity. The region of HIV-1 RT between amino acids 120 and 150 lies within the fingers subdomain of the HIV-1 polymerase. We believe that the fingers subdomain plays a role in positioning the template. Many amino acid substitutions in this region decrease or abolish both the polymerase and the RNase H functions.
Boyer
P L
PL
ABL-Basic Research Program, NCI-Frederick Cancer and Development Center, Frederick, MD 21702-1201.
Ferris
A L
AL
Clark
P
P
Whitmer
J
J
Frank
P
P
Tantillo
C
C
Arnold
E
E
Hughes
S H
SH
D000596:Amino Acids / Q000502:physiology; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology*; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism
D000596:Amino Acids; D011089:Polydeoxyribonucleotides; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H