Minimal core domain of HIV-1 integrase for biological activity.
The human immunodeficiency virus type-1 (HIV-1) integrase (IN) mediates insertion of viral DNA into human DNA, which is an essential step in the viral life cycle. In order to study minimal core domain in HIV-1 IN protein, we constructed nine deletion mutants by using PCR amplification. The constructs were expressed in Escherichia coli, and the proteins were subsequently purified and analyzed in terms of biological activity such as enzymatic and DNA-binding activities. The mutant INs with an N-terminal or C-terminal deletion showed strong disintegration activity though they failed to show endonucleolytic and strand transfer activities, indicating that the disintegration reaction does not require the fine structure of the HIV-1 IN protein. In the DNA-binding analysis using gel mobility shift assay and UV cross-linking method, it was found that both the central and C-terminal domains are essential for proper DNA-IN protein interaction although the central or C-terminal domain alone was able to be in close contact with DNA substrate. Therefore, our results suggest that the C-terminal domain act as a DNA-holding motive, which leads to proper interaction for enzymatic reaction between the IN protein and DNA.
Kim
D J
DJ
Department of Biotechnology, Chung-Ang University, Kyunggido, Korea.
Lee
S K
SK
Oh
Y T
YT
Shin
C G
CG
D001665:Binding Sites / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019427:HIV Integrase / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D019427:HIV Integrase