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. within the SSC, a conclusion that is also in agreement with atomic force microscope images of the stable nucleoprotein complexes. 20bp of terminal viral DNA end sequence are efficient substrates for half-site integration in vitro, and are the only protected region observed in footprinting experiments. However, several hundred base pairs of non-specific flanking DNA sequence are required for efficient SSC assembly, stability and concerted integration. We are probing the function of this non-specific DNA sequence in SSC assembly and stability and propose that non-specific interactions between IN and DNA (distinct from the stable association of a tetramer of IN with the viral DNA ends) are involved. Having established methodologies to assemble synaptic complexes in vitro with purified integrase and HIV-1 DNA substrate, we are attempting both low and high-resolution structural studies combined with biochemical approaches to understand the detailed mechanism of DNA integration. The potential role of cellular proteins in SSC assembly is under investigation. One cellular protein that has been implicated in playing an important role in HIV-1 DNA integration is Lens Epithelial Derived Growth Factor (LEDGF). We find that LEDGF does not stimulate assembly of the SSC and in fact inhibits complex assembly. LEDGF must therefore be acquired by the preintegration complex after the two viral DNA ends are engaged by integrase to form the SSC. Our goal is directed towards X-ray crystallographic studies of HIV-1 intasomes. The two major obstacles we need to overcome are the propensity of the intasomes to self-associate and the requirement of several hundred base pairs of non-specific flanking DNA sequence for their assembly. The role of the flanking DNA is not understood. Mixing experiments with long viral DNA ends, and short viral DNAends that do not assemble intasomes alone, show that the short DNA ends are efficiently incorporated into intasomes if the partner is long. The length requirement of several hundred base pairs is similar to the length required for DNA to be easily able to bend back on itself. Our current hypothesis is that is that the flanking DNA transiently associates with the intasome, perhaps occupying the target DNA binding site. Cherepanov and colleagues have recently solved the structure of the prototype foamy virus (PFV) intasome. PFV belongs to the Spumaretrovirus family. Although it shares only 13% identify with HIV-1 integrase and has an extra domain not found in the HIV-1 protein, the arrangement of domains in the HIV-1 intasome is unlikely to be radically different. However, PFV integrase is sufficiently different that structures of the HIV-1 intasome are required to understand the detailed mechanisms of resistance to inhibitors such as Raltegravir. Four of the domains in the in the PFV intasome structure are disordered. The requirement of several hundred base pairs of internal DNA for HIV-1 intasome assembly and stability does not allow us approach structural studies in the way that has been successful with PFV intasomes. Our first approach was to assemble intasomes with long viral DNA substrate and cut off the internal DNA segment after assembly, but we found that the intasomes dissociate after cleavage of this flanking DNA. However, once target DNA is captured and stand transfer has taken place the internal DNA can be cleaved without loss of stability. A major effort is therefore directed at assembling HIV-1 STCs for structural studies. We are using the approach of assembling STCs on branched DNAs corresponding to the product of integration, a strategy we have successfully applied to the PFV system. We have exploited prototype foamy virus (PFV) as a model system explore novel strategies for obtaining high-resolution structures of HIV-1 intasomes. We have shown that PFV integrase can assemble STCs on product DNA, bypassing the requirement for assembly through the normal reaction pathway. High-resolution structural studies of these complexes shows they are identical to those formed on the normal reaction pathway as previously determined by the Cherepanov group. In addition, we observe additional electron density that likely corresponds to the domains that were missing in the earlier structure. Based on this proof of concept with PFV, we are currently applying a similar strategy to the HIV-1 system. Four of the domains in the PFV integrase structures are disordered. We have tested whether these domains are necessary for function of HIV-1 integrase by constructing heterodimers lacking the corresponding domains. We find that intasomes can be assembled when the outer subunits lack the domains that are missing in the PFV intasome structures. These domains are therefore not essential for function. However stable heterodimers of HIV-1 integraase comprising full-length protein in a dimer with the catalytic domain are inactive and do not assemble intasomes. We propose that that monomeric integrase is an obligatory intermediate on the intasome assembly pathway. Stabilization of the catalytic domain dimer interface therefore blocks intasome assembly. We have engineered a hyperactive mutant of HIV-1 integrase by fusing the DNA binding domain of Sulfolobus solfataricus chromosomal protein (Pdb: 1BNZ) to the N-terminus of integrase. This fusion protein has much higher in vitro concerted activity and, unlike the wild type protein, it is active on oligonucleotide viral DNA substrate. This activity with short DNA substrates overcomes one of the major obstacles to structural studies. In addition, the fusion protein has much better solubility properties than the wild type protein. Intasomes containing the fusion protein have been prepared at concentrations suitable for structural studies. Although these intasomes aggregate much less than those assembled with wild type protein, aggregation is still an issue at the concentrations required for crystallization. We are attempting to identify mutations that further improve the solubility properties of assembled intasomes.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$965,407
Indirect Cost
City
State
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Li, Min; Jurado, Kellie A; Lin, Shiqiang et al. (2014) Engineered hyperactive integrase for concerted HIV-1 DNA integration. PLoS One 9:e105078
Li, Min; Craigie, Robert (2009) Nucleoprotein complex intermediates in HIV-1 integration. Methods 47:237-42