We have established conditions for in vitro assembly of stable synaptic complexes of a pair of viral DNA ends with HIV-1 integrase. These nucleoprotein complexes are intermediates in the integration of HIV DNA into a target DNA. Furthermore, the association of integrase with viral DNA in these complexes mimics all the properties of the association of integrase with viral DNA in preintegration complexes (PICs) isolated from virus infected cells. The synaptic complexes contain a tetramer of integrase tightly bounds to a pair of viral DNA ends. Footprinting of the viral DNA ends within the complex reveals that less than 20 base pairs of terminal viral DNA sequence are protected by integrase.? Assembly of synaptic complexes exhibits a critical dependence on assembly reaction conditions. Under most reaction conditions for integration in vitro, the majority of the reaction products are half-site products that result from integration of only one viral DNA end into one strand of the target DNA. Stable synaptic complexes are not formed under such reaction conditions. However, under conditions that promote concerted integration of pairs of viral DNA ends as occurs in vivo, synaptic complexes are efficiently assembled. Surprisingly, efficient synaptic complex assembly requires the viral DNA substrate to be longer than 300 bp; complex assembly is essentially undetectable with substrates shorter than 100 bp. This is unexpected because once assembled integrase protects less than 20 bp of terminal sequence. Although the complexes are highly stable, even when challenged with greater than 0.5M NaCl, the stability is progressively decreased as the viral DNA is shortened by cleavage with restriction endonucleases. We interpret this to mean that transient interactions with integrase along the length of the viral DNA contribute to the stability of the synaptic complex. Interaction of the viral DNA ends with integrase spanning several hundred base pairs of terminal DNA sequence is consistent with the results of footprinting of PICs formed in virus infected cells. Several hundred base pairs of terminal DNA sequence are protected and this protection is dependent upon the presence of integrase.? We have developed methods to prepare large quantities of synaptic complexes for biochemical studies. In particular, we are collaborating with Dr. Mamuka Kvaratskhelia to use mass spectroscopy in combination with protein footprinting to determine the functional protein-protein interfaces in the synaptic complex and the path of the DNA. We are also collaborating with Kiyoshi Mizuuchi to determine the path of the DNA in the complex by fluorescence resonance energy transfer (FRET).
| Li, Min; Mizuuchi, Michiyo; Burke Jr, Terrence R et al. (2006) Retroviral DNA integration: reaction pathway and critical intermediates. EMBO J 25:1295-304 |
