The X-ray structures of the 4-domain prototype foamy virus (PFV) integrase (IN)(1,2) and very recently the 3-domain Rous sarcoma virus (RSV) IN (3) in complex with viral DNA and/or viral-target DNA substrates have been determined. Yet, the crystal structure of the medically important 3- domain HIV IN by itself or in complex with DNA is still elusive. We propose to provide the means and methods using monomeric wild type (wt) HIV IN or modified IN constructs to accomplish this very difficult but highly significant project. The first necessary step to achieve this important goal is harvesting the ability of HIV IN to assemble these IN-DNA complexes at high protein concentrations in the ~100 to 150 M range for crystal screening and other in-solution structural investigations. Our recent success in defining new assembly pathways for stabilizing 3-domain IN-DNA complexes and the use of newly enhanced solubility methods for recombinant HIV IN will advance our investigative studies.
It is necessary to obtain the 3-dimensional structure of the HIV IN in complex with viral DNA which currently does not exist to better understand the mechanism of integrase inhibitor interactions and drug resistance. We propose to facilitate the isolation of kinetically stabilized HIV IN-DNA complexes at high concentrations in-solution and screening of these complexes for crystal formation. With resolved IN-DNA structures, new allosteric inhibitor sites outside the active catalytic site in the presence of DNA may be identified and possibly provide mechanisms for pre-prophylaxis treatment.
| Pandey, Krishan K; Bera, Sibes; Shi, Ke et al. (2017) A C-terminal ""Tail"" Region in the Rous Sarcoma Virus Integrase Provides High Plasticity of Functional Integrase Oligomerization during Intasome Assembly. J Biol Chem 292:5018-5030 |
