Study On The Development And Applications Of Activity Assays For HIV-1 Integrase

Acquired immunodeficiency syndrome (AIDS), caused by the human immunodeficiency virus (HIV), is one of the most serious and deadly diseases in human history. Right now, there is still no cure for AIDS. HIV-encoded integrase (IN) catalyzes the integration of the viral DNA into the host genome, and IN is one of the vital enzymes necessary for viral replication. The inhibition of IN activities can effectively block the viral replication cycle of HIV in host cells. Moreover, there is no recognized counterpart of IN in normal human cells, and inhibitors specifically targeting IN may thus have little side effect on human body. Therefore, IN is considered to be an ideal target for the research and development of anti-HIV drugs.IN catalyzes two successive reactions, termed 3′-processing and strand transfer, to facilitate the integration process in vivo. Both the above reactions can be modeled in vitro using purified recombinant IN protein, oligonucleotide DNA substrates, and divalent cationic cofactor. Besides, in vitro, IN can also carry out an apparent reversal of strand transfer which has been termed disintegration, in which the DNA product of strand transfer is resolved into viral and target DNA segments. To develop high efficient assays for IN activities and apply these assays to screen inhibitors is the main method for in vitro IN inhibitor screening, and it is also a focus in the research field of antiviral drug development targeting IN. In this thesis, different types of recombinant IN proteins are expressed and purified, and a series of high-throughput assays for IN activities have been developed. These assays have also been applied in the studies of the properties of IN and IN inhibitor screening. The main content of the thesis consists of the following major aspects:(1) Expression, purification of integrase protein and development of a high-throughput assay for the 3′-processing reaction of integraseThe IN gene of HIV HXB2CG strain was acquired by PCR. After sequence analysis, primers for gene mutation were designed and site-directed mutagenesis of the HXB2CG IN gene was done by overlapping PCR to construct the IN gene of HIV NL4-3 strain. Site-directed mutagenesis was also employed to bring the F185K/C280S mutations to the constructed NL4-3 IN gene for the purpose of enhancing the protein solubility. The correctly constructed IN gene was ligated to a pET expression plasmid vector, and IN was highly expressed as a soluble protein in Escherichia coli strain BL21. After purification from the supernatant of cell suspension using affinity chromatography, the highly purified recombinant IN protein was active in 3′-processing and strand transfer reactions.Based on the principle of molecular beacons, we designed a fluorophore and a quencher labeled 3′-processing DNA substrate mimicking the viral DNA and developed a novel fluorescent assay for the detection of IN 3′-processing activity. The results obtained show that this assay is an overall liquid assay with high sensitivity, high specificity, easy and simple assay procedure, as well as real-time monitoring of the 3′-processing reaction of IN. The assay is also applicable in the 3′-processing reaction character study of IN and high-throughput screening of inhibitors targeting the 3′-processing reaction of IN. The fluorescent assay for IN 3′-processing reaction proposed in this thesis has the potential to be applied in the studies of interactions between other proteins and DNA. This work has important academic significance and application value.(2) Development and application of a high-throughput assay for the strand transfer reaction of integraseThe inhibition of strand transfer reaction is reported to be the primary key to block the biological functions of IN in vivo. Nowadays, In vitro assays for IN inhibitor screening are generally based on the strand transfer reaction. In this work, biotin and digoxin modified donor DNA and target DNA were designed and composed, and streptavidin-coated magnetic beads were involved to capture the reaction product DNA strand, and a novel high-throughput enzyme-linked immunosorbent assay (ELISA) was developed to measure the IN-catalyzed strand transfer reaction activity or 3′-processing and strand transfer reaction activities altogether. Compared to previous assays, the high-throughput ELISA proposed in this thesis has notable improvements: (i) It is a high-throughput format assay with no need of radioactive substrate; (ii) All the reagents are freely suspended in solution, makes the magnetic beads–DNA product contact and subsequent magnetic beads–antibody contact much easier and more sufficient, the sensitivity of this assay is thus enhanced. In addition, the assay is flexible to investigate the interactions among all reagents and it is easy to study the mechanism of IN inhibitors; (iii) Neither the precoating nor the blocking of microplate is required, it is less laborious and time consuming. The easy transfer of magnetic beads into fresh microplate before detection helps to eliminate almost all the non-specific binding of reagents on the microplate, the background readings are effectively reduced and the higher specificity of this assay is thus achieved; (iv) Both the ELISA and the fluorescence-linked immunosorbent assay (FLISA) associated detection strategy are used, and the applicable range of this assay is expanded.The high-throughput ELISA was successfully applied to study the effects of divalent cations on the strand transfer reaction, and several meaningful results were obtained. The assay was also proved to be effective in inhibitor identification of IN by the employment of known IN inhibitors. Several samples from natural product extracts and composed compounds were screened out to be active IN inhibitors by using this high-throughput ELISA.(3) Development of a high-throughput assay for the disintegration reaction of integraseIN can carry out the disintegration reaction in vitro, a reversal of the integration process. The central catalytic domain of integrase (IN-CCD) is capable of catalyzing disintegration reaction alone. To develop high-throughput assays for disintegration reaction is of great significance for investigating the functions of IN and IN inhibitor screening. In this work, wild type IN and IN-CCD proteins with functional activities were expressed and purified. Biotin and digoxin-labeled disintegration DNA substrate was designed and composed. Based on the application of streptavidin-coated magnetic beads to capture the biotin-labeled DNA, we proposed a high-throughput ELISA for detecting the disintegration activity of IN and IN-CCD in vitro.We studied the effects of metal ions on the disintegration reaction using this high-throughput disintegration ELISA. The results showed that as in 3′-processing and strand transfer, IN displayed dramatic preference for Mn2+ over Mg2+ to be the cationic cofactor in disintegration. Further NaCl titration study indicates that the preference for Mn2+ over Mg2+ in disintegration reaction is ascribed to a higher effect of Mn2+ than Mg2+ in stabilizing the IN–DNA complex. Baicalein, a known IN inhibitor, was involved to test the efficiency of this high-throughput disintegration ELISA in IN inhibitor screening. The results proved that baicalein clearly inhibit IN activities by targeting IN-CCD protein. The high-throughput disintegration ELISA presented in this work has the advantages of high-throughput, high sensitivity, high specificity, low background, as well as less laborious and time consuming. The assay is capable to be applied to study the disintegration reaction character and pharmacology of IN inhibitors. In addition, the assay has the potential to be applied for the high-throughput identification of drug candidates targeting IN, especially targeting IN-CCD.(4) Study on the activity and solubility of the wild type and F185K soluble mutant type integrase central catalytic domainDue to their poor solubility, the wild type (WT) IN and IN-CCD proteins form insoluble inclusion bodies when expressed in Escherichia coli, which brings difficulty in subsequent purification and functional studies. The introduction of F185K mutation into IN gene enhances the solubility of IN, and both IN and IN-CCD can be expressed as soluble protein, whereas the activities of IN and IN-CCD are not affected. In this work, the WT and F185K mutant type IN-CCD proteins were expressed and purified, and their solubility and activity were compared. The solubility and activities of WT and F185K/C280S full IN proteins were also compared. The results show that after F185K and F185K/C280S mutations, the solubility of IN-CCD and full IN proteins were both dramatically increased, both proteins were expressed as soluble proteins. In the meantime, the disintegration activity of mutant type IN-CCD, and the 3′-processing and strand transfer activities of mutant type full IN were reduced to some extent.We further constructed the WT and F185K mutant type IN-CCD structures by homology modeling, and 1800 ps of molecular dynamics (MD) simulations for these two types of IN-CCD proteins in water were performed. Some meaningful results were obtained: (i) After the F185K mutation, the flexibility of the catalytic loop region and the total mobility of IN-CCD was reduced, whereas the distances between the residues of the catalytic site (DDE motif) had no notable change. Therefore, the activities of mutated proteins were decreased, but were not significantly affected. (ii) After the F185K mutation, changes of the electrostatic interaction network drove the conformational change of IN-CCD, especially changed the conformation of the loop regions, and resulted in the burying of some hydrophobic residues and exposure of some other hydrophilic residues on the protein surface. The relative hydrophilic solvent accessible surface area of IN-CCD was increased. Moreover, the F185K mutation notably increased the hydrogen bonds between the IN-CCD protein and water molecules. These above changes contribute to the solubility increase of IN-CCD. It is found that the results obtained from MD simulations are in good agreement with the experiment data. This work supplies useful information and provides valuable insight for understanding the protein solubility and will be helpful in protein engineering for increasing the solubility of proteins.