Retroviral DNA integration directed by HIV integration protein in vitro.
Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a lambda DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.
Bushman
F D
FD
Laboratory of Molecular Biology, National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Fujiwara
T
T
Craigie
R
R
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002474:Cell-Free System; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004251:DNA Transposable Elements; D004279:DNA, Viral / Q000235:genetics*; D006678:HIV / Q000201:enzymology* / Q000235:genetics; D007304:Insect Viruses / Q000235:genetics; D019426:Integrases; D009052:Leukemia Virus, Murine / Q000235:genetics*; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes; D015183:Restriction Mapping
D004251:DNA Transposable Elements; D004279:DNA, Viral; D015345:Oligonucleotide Probes; D004254:DNA Nucleotidyltransferases; D019426:Integrases