Biochemical characterization of recombinant equine infectious anemia virus integrase.
The integrase from equine infectious anemia virus (ELAV) was expressed in Escherichia coli as a polyhistidine fusion protein. The protein was purified under native and denaturing conditions using one-step nickel-affinity chromatography. The purified denatured protein was refolded in the presence of detergent. In vitro 3' processing and DNA strand transfer activities were analyzed under Mg(2+)- and Mn(2+)-dependent reaction conditions. Both protein preparations were similarly active. Only one viral DNA end was efficiently integrated during Mn(2+)-and Mg(2+)-dependent DNA strand transfer. Water was the predominant nucleophile for Mg(2+)- and Mn(2+)-dependent 3' processing activity. The results underscore functional similarities between EIAV integrase and the previously characterized HIV-1 enzyme.
Engelman
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Division of Human Retrovirology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.
D002846:Chromatography, Affinity; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006639:Histidine / Q000235:genetics; D004860:Infectious Anemia Virus, Equine / Q000201:enzymology* / Q000235:genetics; D019426:Integrases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016662:Virus Integration; D014867:Water
D004279:DNA, Viral; D011994:Recombinant Proteins; D014867:Water; D008345:Manganese; D006639:Histidine; D019426:Integrases; D008274:Magnesium