Mutational analysis of Phe160 within the "palm" subdomain of human immunodeficiency virus type 1 reverse transcriptase.
The highly conserved Phe160 residue is located in the "palm" subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), and makes contact with Tyr115, a residue which is involved in deoxynucleoside triphosphate (dNTP) binding and fidelity of DNA synthesis. Five mutant RTs having Tyr, Trp, Ile, Ala or Gln instead of Phe160 were obtained by site-directed mutagenesis. F160Y and F160W retained substantial DNA polymerase activity, whereas the catalytic efficiency of nucleotide incorporation of mutants F160I, F160A and F160Q was less than 10 % that of the wild-type RT, using poly(rA).oligo(dT)20 as the template-primer. The low catalytic efficiency of mutants F160I, F160A and F160Q was due to their lower affinity for the dNTP substrate. F160Y displayed similar kinetic parameters as the wild-type RT in nucleotide insertion assays carried out with heteropolymeric DNA/DNA template-primers. However, nucleotide affinity was two- to sixfold reduced in the case of mutant F160W. Fidelity assays revealed similar misinsertion and mispair extension ratios for the three enzymes, although F160W showed a slightly higher accuracy of DNA synthesis, particularly in the presence of high concentrations of dNTP. When introduced in an infectious proviral clone, mutations F160I, F160A and F160Q rendered non-viable virus. The importance of Phe160 for polymerase function and viral replication could be mediated by its interaction with Tyr115, as suggested by the analysis of the available crystal structures of HIV-1 RT.
Centro de Biología Molecular "Severo Ochoa", CSIC-Universidad Autónoma de Madrid, Cantoblanco 28049, Spain.
M
M
Gutiérrez-Rivas
A
A
Ibáñez
M A
MA
Martínez
E
E
Domingo
L
L
Menéndez-Arias
D019943:Amino Acid Substitution; D001483:Base Sequence; D018360:Crystallography, X-Ray; D004247:DNA; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011995:Recombination, Genetic; D014779:Virus Replication
D010649:Phenylalanine; D004247:DNA; D054303:HIV Reverse Transcriptase