DNA requirements for assembly and stability of HIV-1 intasomes
Open Access
- 31 January 2012
- journal article
- research article
- Published by Wiley in Protein Science
- Vol. 21 (2), 249-257
- https://doi.org/10.1002/pro.2010
Abstract
Integration of viral DNA into the host genome is an essential step in retroviral replication that is mediated by a stable nucleoprotein complex comprising a tetramer of integrase bridging the two ends of the viral DNA in a stable synaptic complex (SSC) or intasome. Assembly of HIV-1 intasomes requires several hundred base pairs of nonspecific internal DNA in addition to the terminal viral DNA sequence that is protected in footprinting experiments. We find that only one of the viral DNA ends in the intasome requires long-nonspecific internal DNA for intasome assembly. Although intasomes are unstable in solution when the nonspecific internal DNA is cut off after assembly, they are stable in agarose gels. These complexes are indistinguishable from SSCs with nonspecific internal DNA in Forster resonance energy transfer (FRET) experiments suggesting the interactions with the viral DNA and integrase tetramer are the same regardless of the presence of nonspecific internal DNA. We discuss models of how the internal DNA contributes to intasome assembly and stability. FRET is exquisitely sensitive to the distance between the fluorophores and given certain assumptions can be translated to distance measurements. We anticipated that a set of such distance constraints would provide a map of the DNA path within the intasome. In reality, the constraints we could impose from the FRET data were quite weak allowing a wide envelope for the possible path. We discuss the difficulties of converting the FRET signal to absolute distance within nucleoprotein complexes.Keywords
Funding Information
- Intramural Program of NIDDK, NIH
- AIDS Targeted Antiviral Program of the Office of the Director of the NIH
This publication has 11 references indexed in Scilit:
- Nucleoprotein Intermediates in HIV-1 DNA Integration Visualized by Atomic Force MicroscopyJournal of Molecular Biology, 2010
- Retroviral intasome assembly and inhibition of DNA strand transferNature, 2010
- Impact of Emission Anisotropy on Fluorescence Spectroscopy and FRET Distance MeasurementsBiophysical Journal, 2009
- Molecular Interactions between HIV-1 Integrase and the Two Viral DNA Ends within the Synaptic Complex that Mediates Concerted IntegrationJournal of Molecular Biology, 2009
- Architecture of the 99 bp DNA-Six-Protein Regulatory Complex of the λ att SiteMolecular Cell, 2006
- Tn10 transpososome assembly involves a folded intermediate that must be unfolded for target capture and strand transferThe EMBO Journal, 2000
- IHF Modulation of Tn10 Transposition: Sensory Transduction of Supercoiling Status via a Proposed Protein/DNA Molecular SpringCell, 1998
- HIV-1 cDNA Integration: Requirement of HMG I(Y) Protein for Function of Preintegration Complexes In VitroCell, 1997
- Protection of retroviral DNA from autointegration: involvement of a cellular factor.Proceedings of the National Academy of Sciences, 1994
- A nucleoprotein complex mediates the integration of retroviral DNA.Genes & Development, 1989