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        Our group has two proteins (AnkGAG1D4 and 2LTRZFP) having been proved that they are able to inhibit a HIV-1 replication. Knowledge of immunology, molecular biology, and computational chemistry is used to reveal their function. Both proteins are aimed to cure the HIV-infected patient by gene therapy. To illustrate the past and future of our working, the research is classified and described what they are studied.
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Proteins:
Enhancing binding affinity:
Studying mechanism:
Stem cells:
Animal model:
 
     
 
 
  AnkGAG1D4  
 
        An ankyrin with three internal modules named AnkGAG1D4 have been proposed that it interrupts an HIV-1 replication at the step of gag polymerization.ref AnkGAG1D4 was provided by constructing a phage-displayed library and screening on a fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. Our ankyrin consensus library, based on DARPins librariesref with the different modifications are shown in Fig.1A, allows to randomizing on the variable residues (positions 2, 3, 5, 10, 13 and 14; Fig. 1B) of ankyrin which is accessible surface interacted to desired target. Meanwhile the structure is not destroyed because of the high stability of conserve residues. The 3D structure of AnkGAG1D4 was solved by X-ray crystallography as shown in Fig. 1C and already deposited in PDB (PDBid: 4HLL)ref.
     
        The effectiveness of AnkGAG1D4 was shown that the HIV viral load and CA (or p24) level are significantly reduced in SupT1 cells expressing myristoylated AnkGAG1D4 (Myr+AnkGAG1D4) and non-myristoylated AnkGAG1D4 (Myr0AnkGAG1D4) comparing to control (Fig. 2A and 2B). Noted, AnkA32D3 is negative binder to HIV-1 Gag precursor.
Figure 1 Ankyrin consensus sequence and 3D structure of AnkGAG1D4. (A), Sequence comparison between DARPin libraryref and our library. The red and black alphabet are variable and conserve residues, respectively, and blue is differrent modication derived from DARPin. (B), Single modue of ankyrin representing variable residues. (C) 3D structure of AnkGAG1D4 (PDBid = 4HLL)

        Confocal microscopy illustrated that, at day 11 after HIV infection, the AnkGAG1D4 interact to the Gag in both the cytoplasm and the plasma membrane as shown in Fig. 2C. Moreover, a stoichiometry (N) analyzed by microcalorimetry (ITC) of the pair AnkGAG1D4/H6MA-CA (N = 0.91) implies that AnkGAG1D4 binds to the H6MA-CA monomer in a 1-to-1 binary complex with the binding affinity in micromolar level (Kd ~ 1 µM).      Target of AnkGAG1D4 is N-terminal domain of CA (NTDCA) characterized by testing CA residues 1-110 and AnkGAG1D4 in ELISA. Interesting, Ankyrinotope shows that AnkGAG1D4 binds to helices 1 or 7 ofNTDCA in which relatives with mutagenesis results showed that R18ACA (helix 1), R132ACA (helix 7), and R143ACA (helix 7) reduce binding affinity to AnkGAG1D4.

 
Figure 2 Inhibitory effect of ankyrins on HIV-1 replication. (A), the CA level in culture supernatants collected at time intervals (5, 7, 9, 11 and 13 days post HIV infection, using ELISA. (B), Viral load in the culture supernatnats collected on day-11, using Cobas Amplicor tests. (C), Confocal microscopy of HIV-1 infected SupT1 expressing ankyrins collected at day 11 post infection. Merged images are enlarged and shown on the right side of eahc panel. White arrows point the colocalization of ankyrin and Gag. R, the Pearson correlation coefficient for signal colocalization.
 
     
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  2LTRZFP  
 
        2LTRZFP is a zinc finger protein (ZFP) which was designed to recognize at the end terminal part of HIV LTR which is the target area of integrase enzyme. This ZFP is based on Cys2His2 ZFP which is commonly bound to specific DNA in the human and metazoan genome. General ZFP is composed by a motif of a ββα molecule in 30 residues length. The recognize size of ZFP to DNA on 3 contiguous base pair of major groove is the positions -1, 3, and 6.
Figure 3 Schematic representation of the circle junction region of HIV LTRref. The sequence in box was used to desing the 2LTRZFP.

        Our ZFP was initially designed by submitting the DNA sequence of the circle junction region of HIV LTR in ZF Tool server. The ZF Tool provided the sequence of 5’ CTAGCAGTACTGGATGGG 3’ which is the top score for designer the ZFP. Then, the tool generated the 176 full-length amino acids for a six ββα motif. Each motif was linked with the canocical TGEKP linker. This 2LTRZFP was fused to GFP, called 2LTRZFP-GFP, at its C-terminal region. Under microscopy, the green fluorescence from GFP is observed in the nuclease and also cytoplasm of mamalian cell. This confirms that it folds correctly in cytoplasm and homes to the nucleas.

        The binding kinetic between 2LTRZFP-GFP and its target DNA was determined by the method of surface plasmon resonance (SPR). The result showed that the binding affinity of 2LTRZFP-GFP to target DNA is in a nanomolar scale with Kd = 12.0 nM. The effective of 2LTRZFP was demonstrated that 2LTRZFP protein interferes the integrases activity during the joining of HIV DNA to host DNA by determining p24 level and Alu-gag qPCR. The result showed that the p24 level cannot be detected in cell expressing 2LTRZFP at day 13 after virus challenging (Fig. 4a) and the protection expand up to 17 days postchalenge (Fig. 4B). Moreover, the extracted genomic DNA on day 13 after challenge was not observed in cell presenting 2LTRZFP by Alu-gag qPCR (Fig. 4c).

 
Figure 4 Inhibition of HIV-1 replication in SupT1 cells in SupT1 cells stably expressing 2LTRZFP-GFP. (A), p24 level in supernatants at day 13 after HIV virsu challenge. (B), p24 level in supernatants in cells infected 5 MOI. (C), the Alu-gag qPCR of extracted genmomic DNA from infected cell at day 13 after challenge.
 
     
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  Role of AnkGAG1D4 and 2LTRZFP in HIV life cycle  
 
        Our drug proteins are designed by interupting the steps during the retrovirus life cycle, unlike the direct binding of the drug to the viral particles. For example, AnkGAG1D4 and 2LTRZFP interfere the HIV replication at the steps of gag polymerization and integration, respectively. Therefore, awareness the HIV life cycle is inevitably crucial for more understand the drug mechnism and also constructing new drug development.
HIV life cycle
The first step is binding and fusion, gp120 on HIV binds to CD4 and co-receptor (CCR5 or CXCR4) on CD4+ cells (Fig. 5(1a)). Then, virus fuses into the host cell (Fig. 5(2a)).
 
Secondly, HIV genome and its proteins are released into the host cell.
 
Reverse transcription, Fig. 5(3a), subsequently occurs when a single-stranded HIV RNA is converted into double- stranded DNA by HIV reverse transcriptase.
 
The HIV DNA is assembled with viral protein (i.e. Vpr, matrix and integrase) and host protein to form as large complex called pre-integration complex (PIC) before entering to nucleas, Fig. 5(4a).
 
The fourth step, called integration, proceeds when an HIV DNA (called unintegrated viral DNA) enters into the nucleus and is integrated with host’s genomic DNA by HIV integrase enzyme (Fig. 5(6a)). There are three forms of unintegrated viral DNA consisting of (i) a linear form which is flanked by long terminal repeat (LTR), (ii) a circular form containing one LTR, and (iii) a circular molecule with two LTRs as shown in Fig. 6. Integrase is involved to join HIV DNA to host DNA by removing 2 bp from the 3' ends of viral DNA (called 3' processing) and inserting both 3' ends of HIV DNA to host DNA.
 
Figure 5 Schematic representation of HIV-1 life cycle. 1a, the HIV infection begins with the binding of gp120 of HIV and CD4 of host cell. 2a, the HIV particle fuses to host membrane. 3a, Reverse transcription. 4a, PIC formation. 5a, PIC enters to host necleas. 6a, Integration. 7a, HIV transcription. 8a, HIV translation. 9a, HIV assemble. 10a, HIV budding.
 
Figure 6 A characteristic of HIV cDNA.
The next step, HIV DNA is then transcribed and translated by the host machineries to yield viral mRNA and viral proteins when the host cell receives a signal to become active (Fig. 5(7a-8a)).
 
In the viral assembly step, the new HIV proteins and viral RNA genome move to host cell membrane and bud out from the host cell to generate an immature HIV (Fig.5(9a-10a)). The imporportant HIV component in this step is Gag protein consisted of of matrix (MA or p17), capsid acid (CA or p24), nucleocapsid (NC), two small spacer peptide (SP1 and SP2), and p6, from N to C terminus. the Gag-Gag interaction and assembly called Gag polymerization is influenced to becoming the HIV assembly.
 
The last step, the HIV proteins are then cleaved by HIV protease and rearranged within HIV particle to become a mature HIV.
Role of AnkGAG1D4 and 2LTRZFP in HIV life cycle
 
These videos are the representation of AnkGAG1D4 and 2LTRZFP role in HIV life cycle. In case of AnkGAG1D4, it binds the NTDCA of Gag leading to interfering Gag polymerization. The result of interuption leads to be unable to export the HIV components. For 2LTRZFP, the hydrophobic interaction between 2LTRZFP and target HIV DNA effects to prevent the binding of integrase to 2LTR of HIV DNA. Therefore, the HIV DNA is not utilized for producing HIV genome and proteins.
HIV life cycle  
AnkGAG1D4 in HIV life cycle 2LTRZFP in HIV life cycle
 
     
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  Enhancing Binding Affinity  
          @ Mutagenesis the AnkGAG1D4  
 
        To improve the potency of AnkGAG1D4 binding, we aim to construct the new version of AnkGAG1D4 which ameliorate binding affinity against HIV-1 capsid proteins (CA) guided by computational approaches. The site-directed mutagenesis has been used to generate mutant AnkGAG1D4. The mutant AnkGAG1D4 protein will further test against CA to monitor their binding affinity. Moreover, crystallography and computational analysis will be carried out to analyze the function of key amino acids in mutant AnkGAG1D4-capsid complex.  This study will lead to produce new-generation AnkGAG1D4 conferred greater protection against HIV-1 infection.
 
     
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          @ Structural bioinformatic  
 
        In order to guide the mutant residues on AnkGAG1D4, the previous AnkGAG1D4-CA complexes will be performed to identify the key player residues on AnkGAG1D4 to CA. Each position of the key players has been calculated the interaction energy i.e. Van der Waal on the minimized complexes in the versions of wild type and mutated residues. Any mutated residue on AnkGAG1D4 provided higher interaction energy than wildtype residue will be predicted for improve binding affity of them.
 
     
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          @ Bi-specific proteins  
          Bi-specific protein is one of application using for expanding the funtional activity of single protein. Its structure is composed of two different effector function or recognition site. This model will be applied with the AnkGAG1D4 to construct the dimers of AnkGAG1D4 or the AnkGAG1D4 conjugated with other effector.  
     
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  Studying Mechanism  
          @ Ankyrinotope  
 
        Ankyrinotope mapping based on competitive ELISA, was used to identify the ankyrin binding area on the NTDCA. For the  experiment,  the CA  was  the  immobilized  ligate,  the AnkGAG1D4 was  the  soluble  ligand,  and  the overlapping  pentadecapeptides (with  a  11-residue  overlap)  belonging  to NTDCA   were  the ligand competitors. The result showed that the helices 1 (H1) and 7 (H7) of NTDCA were investigated to be binding area to AnkGAG1D4 because the pentadecapeptides belonging to helices 1 and 7 showed > 50% inhibition in ankyrinotope mapping by competitive ELISA  (Fig. 7). Therefore, it sugeested that alpha-helices 1 and 7 of  NTDCA could mediate the formation of AnkGAG1D4-CA complex.
Figure 7 Identification of AnkGAG1D4 binding site using ankyrinotope mapping. The F1-F35 peptides belonged to  NTDCA used as competitors and the CA used as positive control
 
     
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          @ Computational modeling  
 
        The crystal AnkGAG1D4 structure at 2.2 Å resolution (PDBid : 4HLL) (Fig. 1C) was constructed and docked to three different conformational CA structures (Fig. 8A-C) relied on ankyrinotope mapping result. Then, the docking complexes were performed to investigate the binding residues of both CA and AnkGAG1D4 by counting interaction pairs within 5 Å length. The result suggested that the AnkGAG1D4 made a higher frequency complex with CA at helix 1 (38 poses in m1 model; m1H1) more than helix 7 (16 poses in m20 model; m20H7), however, the best complex of them at helix 7 (-30 kcal/mol) was more stable than the best complex at helix 1 (-7.9 kcal/mol) (Fig.xD). The binding residues on CA were identified, on complexes of 38 m1H1 and 16 m20H7, and showed that R18 (H1), R132 (H7) and R143 (H7) are key players to AnkGAG1D4.
Fig. 8 3D structure of CA and docking complexes of AnkGAG1D4-CA. (A-C), The three different orientations of CAref which are m1 (A), m5 (B), and m20 (C) models representing the deeply, moderately, and highly accessible of H7, respectively. (D) The best docking complex of m1H1 (AnkGAG1D4 binds to H1 of m1 model) and m20H7 (AnkGAG1D4 binds to H7 of m20 model).
 
     
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               @ Mutagenesis the p24  
 
          In order to confirm the docking data, the predicted binding residues were further analyzed by AMELIA (Fig. 9A). The result showed that the binding activity of AnkGAG1D4 to all mutants (R18ACA, R132ACA, and R143ACA) decreased (Fig. 9C), especially, almost totally abolished binding of R132ACA and R143ACA. Therefore, we could conclude that (1) helices 1 and 7 of  NTDCA bound to AnkGAG1D4-CA complex, however, the interaction of involving H7 are predicted to be more stable than with H1. (2), R18 in H1 and R132 and R143 in H7 of  NTDCA are found to be the key residues of the AnkGAG1D4-CA interaction.
Figure 9 Model of AMELIA and its results. (A), the principle of AMELIA in which biotinylated-AnkGAG1D4 was immobilized on avidin-coated plate. Then, the CA was incubated in coated plate and monitored by monoclonal antibody. (B) The normalization of CA protein amount using a sandwich ELISA CAp24 detection kit. (C) Influence of R-to-A mutation in helices 1 and 7 in AMELIA system.
 
     
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            @ Protein Expression  
 
        In our lab we have a new bicistronic expression system which is 2A peptide and now we are constructing 2A peptide-based lentiviral vector coexpressing 2LTRZFP-mCherry and AnkGAG1D4-GFP which provides anti-HIV activity in integration and viral assembly processes. The 2A peptide has recently become an alternative to IRES because it is more reliable than the IRES and lead to expression of multiple cistrons at equimolar levels. The 2A sequences are relatively short peptides approximately 20 amino acids long and contains a conserved and functional motif D (Asp-Val/Ile-Glu-X-Asn-Pro-Gly-Pro). The 2A peptide also called a ‘‘self-cleaving’’ peptide because it is able to ‘‘cleave’’ at its own C terminus between the last two amino acids (glycine and proline) through ribosomal skipping during protein translation resulting in the expression of two independent proteins from a single transcription event.
 
 
 
Figure 10 Expression of two molecules of protein with a connector 2A. When translation process is occurring a single protein between 2A will be split up (A) cutting the two proteins apart in a specific position (B).
 
     
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          @ Protein Network  
 
        This technique used in this experiment is Liquid chromatography–mass spectrometry (LC-MS, or alternatively HPLC-MS) which is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry (MS). LC/MS has high sensitivity and we can use only small amount of sample for analysis. In this experiment, we would like to observe protein profile and compare protein expression between SupT1 cell line that stably expressed AnkGAG1D4-EGFP and SupT1 cell line. If there are some proteins that up or down-regulated, we will further study on those proteins whether they are essential for cell function or not.
 
 
     
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          @ Interfering Pathway  
 
        The mechanisms of HIV infection, HIV does not only infect cells by cell-free virus particles, it can also infect cells using direct cell-to-cell HIV transmission pathway. Productive cell-to-cell infection requires interaction between the viral envelope glycoproteins (Env) on the surface of the infected cell and HIV receptors on the surfaces of target cells, leading to the formation of virological synapses. In this experiment, we are going to study whether AnkGAG1D4 can block cell-to-cell contact pathway or not. We will mix HIV-preinfected SupT1 that stably expressed AnkGAG1D4-mcherry with Jurkat-GFP. We will observe cells that express GFP. If there are no cells that have GFP, it indicates that Ank1D4 can block HIV cell-to-cell contact pathway.
 
     
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  Stem Cell  
          @ HSC  
 
        Recently, the advances in potential HIV gene therapies combined with hematopoietic stem cell (HSC)-based therapy, has emerged as a promising direction for curing HIV-1 infection. Due to HSCs are capable for self-renewal and differentiation into multi-lineage hematopoietic cell types, including T-lymphocytes and macrophages that have been shown to serve as the primary targets of HIV-1. Transplantation of modified HSCs containing anti-HIV-1 genes in AIDS patient could produce new immune cells that would resist HIV-1 infection. We trust that applications of our anti-HIV-1 molecular scaffolds, 2LTRZFP and Myr(+)AnkGAG1D4, along with hematopoietic stem cells-based gene therapy would provide a long-life resistance to HIV-1 infected individuals with a single time treatment by without daily drug administration for use as an alternative treatment for HAART-resistant patients in the future.
 
     
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       @ iPSC  
 
     Induced pluripotent stem cell (iPSC) technology has provided a promising therapy by production of patient-specific pluripotent stem cells from adult individual. By developing iPSCs to treat HIV, there is the potential for generating a continuous supply of therapeutic cells for transplantation into the patients. According to the our research, designed zinc finger protein, namely 2LTRZFP, and ankyrin-repeat protein, AnkGAG1D4, have recently been generated with the promising function to inhibit HIV viral replication in early and late stages of infection, respectively. In this study, we aim to apply these two anti-HIV proteins as a HIV based stem cell gene therapy. This approach will provide the potential treatment for reconstituting of a patient immune system and also resist to HIV-1 infection using patient own cells generating by iPSC technology.
 
     
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  Animal Model  
          @ Mice  
          This tool will be used to test the effectiveness of 2LTRZFP and Myr(+)AnkGAG1D4 to HIV replication in animal.  
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