Construction of infectious molecular clones of HIV-1 containing defined mutations in the protease gene.
A DNA clone of HIV-1 containing the full-length infectious viral sequence was cleaved at a unique Nco I restriction site within the viral genome, and DNA fragments containing the 5' and 3' portions of the HIV genome were subcloned into separate plasmid vectors. The 5' 'half-virus' construct was further modified by incorporating a class IIS restriction site, Esp3I, near the 3' end of the protease gene of HIV. This site, in combination with a natural ApaI site near the 5' end of the protease gene, creates a convenient cassette shuttle vector in which the protease coding region can be easily replaced. Recombinant viruses containing protease genes either altered by site-directed mutagenesis or amplified from clinical or laboratory isolates can be reconstructed. The DNA fragment containing the protease gene is first subcloned into the 5' half-virus shuttle vector plasmid. Infectious recombinant virus is subsequently recovered by cotransfecting 5' and 3' half-virus plasmids linearized at their common Nco I sites into mammalian cells. This method was successfully applied to constructing viruses containing various substitutions in protease.
Winslow
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DL
DuPont Merck Pharmaceutical Company, Wilmington, DE 19880-0026.
Anton
E D
ED
Horlick
R A
RA
Zagursky
R J
RJ
Tritch
R J
RJ
Scarnati
H
H
Ackerman
K
K
Bacheler
L T
LT
D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D015263:Deoxyribonucleases, Type III Site-Specific; D005814:Genes, Viral; D005822:Genetic Vectors; D016333:HIV Protease / Q000235:genetics*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D004279:DNA, Viral; C071181:endodeoxyribonuclease Esp3I; D015263:Deoxyribonucleases, Type III Site-Specific; D016333:HIV Protease