Nucleoprotein intermediates in HIV-1 DNA integration visualized by atomic force microscopy.
Integration of HIV-1 (human immunodeficiency virus type 1) DNA into the genome of the host cell is an essential step in the viral replication cycle that is mediated by the virally encoded integrase protein. We have used atomic force microscopy to study stable complexes formed between HIV-1 integrase and viral DNA and their interaction with host DNA. A tetramer of integrase stably bridges a pair of viral DNA ends, consistent with previous analysis by gel electrophoresis. The intasome, composed of a tetramer of integrase bridging a pair of viral DNA ends, is highly stable to high ionic strength that would strip more loosely associated integrase from internal regions of the viral DNA. We also observed tetramers of integrase associated with single viral DNA ends; time-course experiments suggest that these may be intermediates in intasome assembly. Strikingly, integrase tetramers are only observed in tight association with viral DNA ends. The self-association properties of intasomes suggest that the integrase tetramer within the intasome is different from the integrase tetramer formed at high concentration in solution in the absence of viral DNA. Finally, the integration product remains tightly bound by the integrase tetramer, but the 3' ends of the target DNA in the complex are not restrained and are free to rotate, resulting in relaxation of initially supercoiled target DNA.
Kotova
Svetlana
S
Laboratory of Bioengineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA.
Li
Min
M
Dimitriadis
Emilios K
EK
Craigie
Robert
R
D024509:3' Flanking Region; D004278:DNA, Superhelical / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry*; D019427:HIV Integrase / Q000737:chemistry*; D015497:HIV-1 / Q000235:genetics*; D018625:Microscopy, Atomic Force; D009698:Nucleoproteins / Q000737:chemistry*; D009994:Osmolar Concentration; D055503:Protein Multimerization; D016662:Virus Integration
D004278:DNA, Superhelical; D004279:DNA, Viral; D009698:Nucleoproteins; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1