PCR amplification of HIV-1 proteinase sequences directly from lab isolates allows determination of five conserved domains.
HIV-1 replication requires limited proteolysis of gag and gag-pol encoded precursor proteins by a specific viral proteinase (PR). Sequences of 20 different HIV-1 strains were compared in order to determine regions of conservation and variability within the PR gene. Viral strains included: (a) five new ones derived from New Orleans patient isolates, (b) four established ones grown in our laboratory, (c) eight, whose sequences were published in the Los Alamos Data Base (1990), (d) one Ugandan, and (e) two Brazilian isolates. In the first two groups, HIV proviral DNA extracted from infected lymphocytes was grown in tissue culture and directly amplified by PCR using specific primers flanking the PR gene. Amplified DNA was directly sequenced using a modified di-deoxy sequencing procedure. Sequence data showed a 25% variation among the 20 different HIV strains studied at the amino acid level, including 8% nonconservative changes and 17% conservative changes. Moreover, five noncontiguous regions were able to be delineated in which the PR showed no amino acid changes. These areas included amino acids (I) 1-9 (amino terminal sequence); (II) 21-32 (sequence around the active site); (III) 47-56 (top of the flap); (IV) 78-88; and (V) 94-99 (carboxy terminal sequence). Our results are consistent with those obtained from X-ray crystallography studies as well as single site mutational analysis.
Fontenot
G
G
Department of Microbiology, Immunology and Parasitology, Louisiana State University Medical Center, New Orleans 70112-1393.
Johnston
K
K
Cohen
J C
JC
Gallaher
W R
WR
Robinson
J
J
Luftig
R B
RB
D000163:Acquired Immunodeficiency Syndrome / Q000382:microbiology; D000595:Amino Acid Sequence; D001483:Base Sequence; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D005814:Genes, Viral; D016333:HIV Protease / Q000737:chemistry / Q000235:genetics*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011487:Protein Conformation
D004279:DNA, Viral; D016333:HIV Protease