Requirement of active human immunodeficiency virus type 1 integrase enzyme for productive infection of human T-lymphoid cells.
The human immunodeficiency virus type 1 (HIV-1) integrase enzyme exhibits significant amino acid sequence conservation with integrase proteins of other retroviruses. We introduced specific amino acid substitutions at a number of the conserved residue positions of recombinant HIV-1 integrase. Some of these substitutions resulted in proteins which were not able to be purified in the same manner as the wild-type enzyme, and these were not studied further. The remaining mutant enzymes were assessed for their abilities to perform functions characteristic of the integrase protein. These included specific removal of the terminal dinucleotides from oligonucleotide substrates representative of the viral U5-long terminal repeat, nonspecific cleavage of oligonucleotide substrates, and mediation of the strand transfer (integration) reaction. Substitution at position 43, within the protein's zinc finger motif region, resulted in an enzyme with reduced specificity for cleavage of the terminal dinucleotide. In addition, a double substitution of aspartic acid and glutamine for valine and glutamic acid, respectively, at positions 151 and 152 within the D,D(35)E motif region rendered the integrase protein inactive for all of its functions. The introduction of this double substitution into an infectious HIV-1 provirus yielded a mutant virus that was incapable of productively infecting human T-lymphoid cells in culture.
LaFemina
R L
RL
Merck Research Laboratories, West Point, Pennsylvania 19486.
Schneider
C L
CL
Robbins
H L
HL
Callahan
P L
PL
LeGrow
K
K
Roth
E
E
Schleif
W A
WA
Emini
E A
EA
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000201:enzymology / Q000472:pathogenicity / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D019426:Integrases; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011533:Proviruses / Q000201:enzymology / Q000472:pathogenicity / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes / Q000382:microbiology*
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D004254:DNA Nucleotidyltransferases; D019426:Integrases