Dimeric Ankyrin with Inverted Module Promotes Bifunctional Property in Capturing Capsid to Impede HIV-1 Replication

Several anti-HIV scaffolds have been proposed as complementary treatments to highly active antiretroviral therapy. AnkGAG1D4, a designed ankyrin repeat protein, formerly demonstrated anti-HIV-1 replication by interfering with HIV-1 Gag polymerization. However, the improvement of the effectiveness was considered. Recently, the dimeric molecules of AnkGAG1D4 were accomplished in enhancing the binding activity against HIV-1 capsid (CAp24). In this study, the interaction of CAp24 against the dimer conformations was elucidated to elaborate the bifunctional property. The accessibility of the ankyrin binding domains was inspected by bio-layer interferometry. By inverting the second module of dimeric ankyrin (AnkGAG1D4NC-CN), the CAp24 interaction KD was significantly reduced. This reflects the capability of AnkGAG1D4NC-CN in simultaneously capturing CAp24. On the contrary, the binding activity of dimeric AnkGAG1D4NC-NC was indistinguishable from the monomeric AnkGAG1D4. The bifunctional property of AnkGAG1D4NC-CN was subsequently confirmed in the secondary reaction with additional p17p24. This data correlates with the MD simulation, which suggested the flexibility of the AnkGAG1D4NC-CN structure. The CAp24 capturing capacity was influenced by the distance of the AnkGAG1D4 binding domains to introduce the avidity mode of AnkGAG1D4NC-CN. Consequently, AnkGAG1D4NC-CN showed superior potency in interfering with HIV-1 NL4-3 WT and HIV-1 NL4-3 MIRCAI201V replication than AnkGAG1D4NC-NC and an affinity improved AnkGAG1D4-S45Y.


Introduction
Non-immunoglobulin scaffolds have been established as promising alternatives to overcome the limitation of monoclonal antibodies. Protein scaffolds offer architecture, lack of disulfide, high solubility, and differential molecular weight, allowing for unique biophysical and pharmacokinetic properties. Scaffold-based platforms are versatile synthetic binders demonstrating many therapeutic prospects, such as quinoline and its derivatives, demonstrating many therapeutic prospects. Quinoline hybrids, a versatile pharmacophore, are clinically used as an antitumor, anti-inflammatory, and anti-human immunodeficiency inverted Ank GAG 1D4 was retained. In vitro, Ank GAG 1D4 NC-CN harbored double-binding domains. The structure of inverted Ank GAG 1D4 was linked with another ankyrin module to determine dimeric conformity.
backbone torsional angles as the original Ank GAG 1D4 ( Figure 1A,B). However, these am acids did not participate in the crucial helix compartments of the ankyrin architectu Notably, the overall superimposed structure of inverted Ank GAG 1D4 was not significan different from its parent structure since the root-mean-square deviation (RMSD) of alp carbons was at 0.2 Å ( Figure 1C). These findings implied that the binding surface of inverted Ank GAG 1D4 was retained. In vitro, Ank GAG 1D4NC-CN harbored double-binding d mains. The structure of inverted Ank GAG 1D4 was linked with another ankyrin module determine dimeric conformity. Figure 1. Initial structure of ankyrin before creating Ank GAG 1D4 dimers: (A) The structure of original Ank GAG 1D4; (B) Inverted Ank GAG 1D4, which is presented in ribbon style from blue at N-terminus to red at the C-terminus; (C) The superimposed structure between the origi Ank GAG 1D4 (pink) and inverted Ank GAG 1D4 (sky) with the yellow showing binding region, DSIGSTPLHLAAYYG 58; (D) The amino sequence of the flexible linker consists of (G4S)4 linker a extra residues. Blue and red are used for superimposition with the first and second ankyrin m ules, respectively; (E) Two versions of Ank GAG 1D4-linker were obtained from superimposition tween Ank GAG 1D4 and a linker. They are initial structures for molecular dynamics simulation. T blue dash line shows how linker distance is measured; (F) Distance of the linker is part of the sim lated dynamics of the Ank GAG 1D4-linker during 5 ns.  4 linker and extra residues. Blue and red are used for superimposition with the first and second ankyrin modules, respectively; (E) Two versions of Ank GAG 1D4-linker were obtained from superimposition between Ank GAG 1D4 and a linker. They are initial structures for molecular dynamics simulation. The blue dash line shows how linker distance is measured; (F) Distance of the linker is part of the simulated dynamics of the Ank GAG 1D4-linker during 5 ns.

Ank GAG 1D4 Dimers Exhibit Diverse Molecular Structures
Ankyrin fusion proteins were constructed to validate their binding properties against CAp24. Initially, Ank GAG 1D4, in conjunction with a flexible linker ( Figure 1D) at its C-terminus (the Ank GAG 1D4-linker), was simulated based on molecular dynamics at 5 ns to observe the dynamic behavior of the interdomain linker in ankyrin dimerization. The result for the Ank GAG 1D4-linker ( Figure 1E) revealed a diversifying conformation with the head-to-tail linker distance ranging between 15.0 and 51.9 Å ( Figure 1F). The C-termini of 500 snapshots of molecular dynamics Ank GAG 1D4-linker were extracted from 0-5 ns simulations and connected with Ank GAG 1D4 to generate the second module of Ank GAG 1D4 NC-NC . Using a similar procedure, these Ank GAG 1D4-linkers were assembled with an inverted Ank GAG 1D4 to obtain Ank GAG 1D4 NC-CN . The majority of the Ank GAG 1D4-linkers (403 of 500) displayed nonoverlapping coordinates with the second ankyrin module in both Ank GAG 1D4 NC-NC and Ank GAG 1D4 NC-CN . The active binding sites of Ank GAG 1D4 NC-CN (71.9 ± 15.0 Å) were more separate than Ank GAG 1D4 NC-NC (59.3 ± 7.5 Å) (Figure 2A). The ratio of ankyrin dimers to CAp24s interaction depends on the distance between binding domains. The distance of 1:1 and 1:2 interactions were 54.8 ± 9.1 Å and 69.7 ± 12.6 Å, respectively ( Figure 2B). Moreover, the conformational variability of the modeled dimers was readily apparent from the exemplifications ( Figure 3). A variety of distances of Ank GAG 1D4 NC-NC and Ank GAG 1D4 NC-CN ranged from long ( Figure 3A,C), medium ( Figure 3C,I) to short ( Figure 3E,K). Interestingly, with the same topology as the Ank GAG 1D4-linker, the direction of the binding surface on the second ankyrin module of Ank GAG 1D4 NC-NC and Ank GAG 1D4 NC-CN differed. For example, the Ank GAG 1D4 NC-NC allowed the two binding sites to face in the same direction ( Figure 3A,C) in a "clam-shaped structure" conformation. However, the binding surfaces of Ank GAG 1D4 NC-CN turned in the opposite direction ( Figure 3G,I). The "clam-shaped structure" conformation was found in Ank GAG 1D4 NC-NC and Ank GAG 1D4 NC-CN ( Figure 3E,K). Conversely, the opposite direction of the binding domain was observed for Ank GAG 1D4 NC-NC .
sult for the Ank GAG 1D4-linker ( Figure 1E) revealed a diversifying conformation with t head-to-tail linker distance ranging between 15.0 and 51.9 Å ( Figure 1F). The C-termini 500 snapshots of molecular dynamics Ank GAG 1D4-linker were extracted from 0-5 ns si ulations and connected with Ank GAG 1D4 to generate the second module of Ank GAG 1D4 NC. Using a similar procedure, these Ank GAG 1D4-linkers were assembled with an invert Ank GAG 1D4 to obtain Ank GAG 1D4NC-CN. The majority of the Ank GAG 1D4-linkers (403 of 5  displayed nonoverlapping coordinates with the second ankyrin module in bo  Ank GAG 1D4NC-NC and Ank GAG 1D4NC-CN. The active binding sites of Ank GAG 1D4NC-CN (71. 15.0 Å) were more separate than Ank GAG 1D4NC-NC (59.3 ± 7.5 Å) (Figure 2A). The ratio ankyrin dimers to CAp24s interaction depends on the distance between binding domai The distance of 1:1 and 1:2 interactions were 54.8 ± 9.1 Å and 69.7 ± 12.6 Å, respectiv ( Figure 2B). Moreover, the conformational variability of the modeled dimers was read apparent from the exemplifications (Figure 3). A variety of distances of Ank GAG 1D4NC and Ank GAG 1D4NC-CN ranged from long ( Figure 3A,C), medium ( Figure 3C,I) to short (F ure 3E,K). Interestingly, with the same topology as the Ank GAG 1D4-linker, the direction the binding surface on the second ankyrin module of Ank GAG 1D4NC-NC and Ank GAG 1D4 CN differed. For example, the Ank GAG 1D4NC-NC allowed the two binding sites to face in t same direction ( Figure 3A,C) in a "clam-shaped structure" conformation. However, t binding surfaces of Ank GAG 1D4NC-CN turned in the opposite direction ( Figure 3G,I). T "clam-shaped structure" conformation was found in Ank GAG 1D4NC-NC and Ank GAG 1D4 CN ( Figure 3E,K). Conversely, the opposite direction of the binding domain was observ for Ank GAG 1D4NC-NC. The binding sites distance of Ank GAG 1D4NC-NC and Ank GAG 1D4NC-CN show 1:1 interaction 54.8 ± 9.1 Å (n = 217) and 1:2 interaction at 69.7 ± 12.6 Å (n = 583). This data denote mean ± SD. * p < 0.0001 using two-tailed unpaired t-test. The binding sites distance of Ank GAG 1D4 NC-NC with 59.3 ± 7.5 Å (n = 397) and Ank GAG 1D4 NC-CN with 71.9 ± 15.0 Å (n = 403); (B) The binding sites distance of Ank GAG 1D4 NC-NC and Ank GAG 1D4 NC-CN show 1:1 interaction at 54.8 ± 9.1 Å (n = 217) and 1:2 interaction at 69.7 ± 12.6 Å (n = 583). This data denote mean ± SD. **** p < 0.0001 using two-tailed unpaired t-test.

Structural Analysis Showed Functional Binding Property of Ank GAG 1D4 Dimers
Owing to the vdw calculation of the interactive energy generated from two CAp24s, the possibility of ankyrin dimers simultaneously capturing with CAp24s was validated. Slightly over half (5.6%) of the Ank GAG 1D4 NC-NC conformations enabled the simultaneous binding of two CAp24s (Table 1 and Figure 3B,F). In contrast, 44.9% showed overlapping coordinates, resulting in the collision of two CAp24 structures or the hindrance of the binding surface of another module from the previously bound CAp24 ( Figure 3D). These findings suggest a 1:1 ratio of Ank GAG 1D4 NC-NC to CAp24. Since 88.6% of the Ank GAG 1D4 NC-CN conformation demonstrated accessible coordinates, the simultaneous accommodation of two CAp24s was more feasible ( Figure 3H,J). Only 11.4% of Ank GAG 1D4 NC-CN presented a 1:1 interaction.

Bifunctional Module Relies on Dimeric Ank GAG 1D4 NC-CN
The results above supported the distinctive binding activity of Ank GAG 1D4 NC-NC and Ank GAG 1D4 NC-CN analyzed by BLI. The functional modules of monomeric Ank GAG 1D4, Ank GAG 1D4 NC-NC , and Ank GAG 1D4 NC-CN were assessed by BLI with two CAp24 molecules ( Figure 4). The biotinylated H 6 -CAp24 was loaded on the biosensor, followed sequentially by binding with the monomeric, dimeric Ank GAG 1D4 NC-NC or Ank GAG 1D4 NC-CN and then the H 6 -p17p24. Ank GAG 1D4 NC-CN bound both CAp24 molecules simultaneously, whereas the monomeric Ank GAG 1D4 and Ank GAG 1D4 NC-NC could not bind the CAp24 using the second arm ( Figure 4A). Ank GAG 1D4 NC-NC is very fast off-rate comparable to monomer, indicating weak binder. Corresponding to binding kinetic constants, Ank GAG 1D4 NC-CN (K D < 1.0 × 10 −12 M) is significantly stronger than Ank GAG 1D4 NC-NC (K D = 1.9 × 10 −8 M) ( Table 2). These data confirmed that both binding pockets of the dimeric Ank GAG 1D4 NC-CN were active, while Ank GAG 1D4 NC-NC has only one main binding site with a much weaker second binding site.  is demonstrated by a sandwich assay using bio-layer interferometry. The biotinylated H6-CAp24 was immobilized to the Octet streptavidin biosensors and sequentially probed with the monomeric or dimeric ankyrins. Subsequently, H6-p17p24 was loaded to monitor the secondary interaction signal. This sensorgram is representative of the triplicate experiment.  Subsequently, H 6 -p17p24 was loaded to monitor the secondary interaction signal. This sensorgram is representative of the triplicate experiment. Table 2. Kinetic characterization of monomeric and dimeric Ank GAG 1D4.

Ankyrin Expression Is Shunted to the Plasma Membrane and Does Not Interfere CD4 Expression in SupT1 Cells
According to an in vitro study in bifunctional modules and molecular structure, dimeric ankyrins are promising in improved anti-HIV-1 activity. Thus, intracellular anti-HIV-1 activity was observed in SupT1 cells. SupT1 cells were transduced with VSV-G pseudotyped lentiviral vector, which carries a gene that encodes myristoylated ankyrin with an  Figure 5A). Each ankyrin-expressing cell was sorted at the same EGFP intensity to obtain an equivalence in ankyrin expression. After cell sorting, all ankyrin-expressing SupT1 cells showed EGFP expression under fluorescence microscopy ( Figure 5B). The level of ankyrin expression was further determined by flow cytometry. The mean fluorescence intensity (MFI) of ankyrin-EGFP in SupT1 cells suggested a comparable ankyrin expression in SupT1 cells. MFI of EGFP was 4.83 × 10 4 , 4.53 × 10 4 , 4.53 × 10 4 , and 4.71 × 10 4 in Ank A3 2D3, Ank GAG 1D4-S45Y, Ank GAG 1D4 NC-NC , and Ank GAG 1D4 NC-CN expressing cell ( Figure 5C). Additionally, the percentage of EGPF positive cells in post-sort Ank A3 2D3, Ank GAG 1D4-S45Y, Ank GAG 1D4 NC-NC , and Ank GAG 1D4 NC-CN expressing cells was 100, 99.8, 99.8, and 99.8%, respectively ( Figure 5D).
Additionally, Ankyrin localization was also determined in HeLa cells by apotome imaging. Ankyrin showed a predominant expression at the plasma membrane and lower distribution in the cytoplasm ( Figure 6A). Apotome imaging suggested that ankyrins were shunted to the plasma membrane, which supports the possibility of ankyrin inhibiting viral replication. Additionally, ankyrin distribution at the plasma membrane was not different in each ankyrin-expressing cell ( Figure 6B).
As a consequence of the N-terminus myristoylated signal, ankyrins were targeted to the inner leaflet of the plasma membrane. Since CD4 is a crucial receptor for HIV-1 entry, it is necessary to determine whether an expression of ankyrin interfered with surface CD4 expression in SupT1 cells. Flow cytometry showed the number of CD4-positive cells in ankyrin-expressing SupT1 to no ankyrin cells ( Figure 7A). MFI of CD4 in SupT1 cells and ankyrin-expressing SupT1 cells (Myr (+) Ank A3 2D3 and Myr (+) Ank GAG 1D4-S45Y, Myr (+) Ank GAG 1D4 NC-NC , and Myr (+) Ank GAG 1D4 NC-CN ) was 3.16 × 10 4 , 3.62 × 10 4 , 3.56 × 10 4 , 4.21 × 10 4 , and 3.57 × 10 4 , respectively ( Figure 7B). These data suggest that myristoylated-ankyrin expression in SupT1 did not interfere with CD4 contents on the cell surface of SupT1 cells.  Additionally, Ankyrin localization was also determined in HeLa cells by apotome imaging. Ankyrin showed a predominant expression at the plasma membrane and lower distribution in the cytoplasm ( Figure 6A). Apotome imaging suggested that ankyrins were shunted to the plasma membrane, which supports the possibility of ankyrin inhibiting viral replication. Additionally, ankyrin distribution at the plasma membrane was not different in each ankyrin-expressing cell ( Figure 6B). As a consequence of the N-terminus myristoylated signal, ankyrins were targeted to the inner leaflet of the plasma membrane. Since CD4 is a crucial receptor for HIV-1 entry, it is necessary to determine whether an expression of ankyrin interfered with surface CD4 expression in SupT1 cells. Flow cytometry showed the number of CD4-positive cells in ankyrin-expressing SupT1 to no ankyrin cells ( Figure 7A). MFI of CD4 in SupT1 cells and ankyrin-expressing SupT1 cells (Myr (+) Ank A3 2D3 and Myr (+) Ank GAG 1D4-S45Y, Myr (+) Ank GAG 1D4NC-NC, and Myr (+) Ank GAG 1D4NC-CN) was 3.16 × 10 4 , 3.62 × 10 4 , 3.56 × 10 4 , 4.21 × 10 4 , and 3.57 × 10 4 , respectively ( Figure 7B). These data suggest that myristoylated-ankyrin expression in SupT1 did not interfere with CD4 contents on the cell surface of SupT1 cells.

Dimeric Ankyrins Improves Antiviral Activity Than Monomeric Ankyrins against HIV-1 Replication
The intracellular anti-HIV-1 activity of dimeric ankyrins was determined in HIV-1 NL4-3 infected SupT1 cells. In this experiment, SupT1 and Ank A3 2D3 expressing SupT1 served as no ankyrin control and irrelevant ankyrin control, respectively. At 7 days post-infection, syncytium cells were observed in SupT1 and Ank A3 2D3 expressing SupT1 ( Figure 8A). On the contrary, Ank GAG 1D4-S45Y, Ank GAG 1D4 NC-NC , and Ank GAG 1D4 NC-CN expressing cells show normal cell morphology until 21 days post-infection. Additionally, the cell viability of infected cells was monitored by CCK-8 assay. SupT1 cell and Ank A3 2D3 expressing SupT1 cells showed a dramatically decreased cell viability at day 19 post-infection ( Figure 8B). The result of SupT1 and Ank A3 2D3 representing cell viability is relevant to cell morphology. In contrast to other ankyrin-expressing cells, cell viability was extended to 21 days.
HIV-1 production in HIV-1 infected SupT1 cells and ankyrin-expressing SupT1 cells was evaluated using HIV-1 p24 ELISA. During culture, the culture supernatant of 7, 13, and 21 days post-infection was harvested and tested for HIV-1 CAp24 level by ELISA. At 7 days post-infection, the level of HIV-1 p24 in the culture supernatant of SupT1 and Ank A3 2D3-expressing SupT1 cells was detected at 1.39 × 10 4 and 1.02 × 10 4 pg/mL ( Figure 9A). Whereas, Ank GAG 1D4-S45Y, Ank GAG 1D4 NC-NC , and Ank GAG 1D4 NC-CN showed a higher potency to suppress HIV-1 propagation. Although Ank GAG 1D4-S45Y suppressed HIV-1 release, their single functional module loss inhibitory activity as a higher CAp24 level was detected on day 21 compared to dimeric ankyrins ( Figure 9B). Interestingly, Ank GAG 1D4 NC-CN showed the highest potency in HIV-1 inhibition. HIV-1 virion packaging was determined as an HIV RNA copy by real-time RT-qPCR. An HIV RNA copy number in Ank GAG 1D4-S45Y, Ank GAG 1D4 NC-NC , and Ank GAG 1D4 NC-CN expressing cells was 1.76 × 10 7 , 6.14 × 10 5 and 3.69 × 10 4 copies/mL, respectively ( Figure 9C). Corresponding to previous findings, the specific interaction of ankyrin to HIV-1 CAp24 interferes with the assembly process leading to a decrease in virion release and disturbing the viral RNA packaging [5]. This finding confers the improvement of anti-HIV-1 activity against HIV-1 NL4-3 WT replication in dimeric ankyrins.

Dimeric Ankyrin Provides Superior Anti-Viral Activity against HIV-1 MIR Virus
The anti-HIV-1 potency of dimeric ankyrins was further investigated in the HIV-1 CAmutation virus. In this study, HIV-1 NL4-3 MIR CAI201V was used as a CA-mutation model. According to cell morphology, no syncytia was observed in HIV-1 NL4-3 MIR CAI201Vinfected cells. However, at day 13 post-infection, SupT1, and Ank A3 2D3-expressing SupT1 cells show a clumping appearance and cell death ( Figure 10A). SupT1 and Ank A3 2D3expressing SupT1 cells show unstable cell viability and tremendously decrease on day 15 post-infection ( Figure 10B). At 7 days post-infection, the CAp24 level in infected SupT1 and Ank A3 2D3 expressing SupT1 cells was detected at 1.58 × 10 3 and 1.80 × 10 3 pg/mL ( Figure 9A). In contrast to other ankyrins, Ank GAG 1D4-S45Y, Ank GAG 1D4 NC-NC , and Ank GAG 1D4 NC-CN exhibited a long-lasting anti-HIV-1 activity as undetectable in CAp24 levels. Nonetheless, this CA-mutation is slow propagation than the WT strain [16,18]. Although CAp24 levels increased in 21 days post-infection, dimeric Ank GAG 1D4 NC-NC and Ank GAG 1D4 NC-CN showed a significant suppression compared with Ank GAG 1D4-S45Y ( Figure 9B). Interestingly, Ank GAG 1D4 NC-CN showed the highest efficiency in inhibiting HIV-1 replication. Real-time RT-qPCR confirmed a higher potency of Ank GAG 1D4 NC-CN due to the lowest HIV-1 RNA copy ( Figure 9C). Although the CAp24 level in Ank GAG 1D4-S45Y expressing cells was slightly different from dimeric ankyrin, RNA copy showed a significantly higher degree than in dimeric ankyrins expressing cells. This data suggested a possibility of aberrant viral core formation, which defects in the viral infectivity of the virions [19]. These results suggested the superior activity of Ank GAG 1D4 NC-CN to inhibit the multiplication of HIV-1 NL4-3 MIR CAI201V virus. Ank GAG 1D4-S45Y, Ank GAG 1D4NC-NC, and Ank GAG 1D4NC-CN expressing cells was 1.76 × 10 7 , 6.14 × 10 5 and 3.69 × 10 4 copies/mL, respectively ( Figure 9C). Corresponding to previous findings, the specific interaction of ankyrin to HIV-1 CAp24 interferes with the assembly process leading to a decrease in virion release and disturbing the viral RNA packaging [5]. This finding confers the improvement of anti-HIV-1 activity against HIV-1 NL4-3 WT replication in dimeric ankyrins.  CA-mutation virus. In this study, HIV-1 NL4-3 MIRCAI201V was used as a CA-mutation model. According to cell morphology, no syncytia was observed in HIV-1 NL4-3 MIRCAI201V-infected cells. However, at day 13 post-infection, SupT1, and Ank A3 2D3-expressing SupT1 cells show a clumping appearance and cell death ( Figure 10A). SupT1 and Ank A3 2D3-expressing SupT1 cells show unstable cell viability and tremendously decrease on day 15 post-infection ( Figure 10B).

Discussion
Designed ankyrin repeat proteins (DARPins) have been selected and focused on therapeutic application. DARPin MP0274, targeting human epidermal growth factor 2 (HER2), has been evaluated in clinical trials for cancer treatment [20]. Improving the binding affinity of DARPins could enhance antiviral activity. Trimeric DARPin, Ensovibep, was explicitly designed to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It includes three individual DARPin domains neutralizing against trimeric SARS-CoV-2 spike protein to reduce viral replication in the lower and upper respiratory tract [21]. Ank GAG 1D4-S45Y mutant as a specific CAp24 domain inhibitor of HIV-1 Gag was constructed using sited-direct mutagenesis. The binding affinity of Ank GAG 1D4-S45Y mutant (KD = 45 nM) was partly enhanced and inhibited either HIV-1 wild-type or the HIV maturation inhibitor-resistant strain for intracellular activity compared to parental Ank GAG 1D4 (KD = 109 nM) [6,7]. Dimeric Ank GAG 1D4 was generated to further improve its binding activity and evaluated for the binding activity [11] ( Figure S1). The (G 4 S) 4 linker was applied to the dimeric ankyrin structure, providing flexibility and high solubility of molecules [22]. The G 4 S linker provides flexibility and solubility in bidomain molecules, such as scFSH scaffolds [23], and αRep homo-bidomain (A3_A3) [24]. For these reasons, the (G 4 S) 4 linker might support the intracellular activity of dimeric ankyrin.
Regarding the BLI and MD, Ank GAG 1D4 NC-NC and Ank GAG 1D4 NC-CN show different binding characteristics. Bio-layer interferometry demonstrated that Ank GAG 1D4 NC-CN remarkably promoted the binding activity, achieving the pM avidity characteristic. The secondary interaction signal of Ank GAG 1D4 NC-CN in BLI suggested the bifunctional motif in contrast to Ank GAG 1D4 NC-NC . In contrast, the binding activity of Ank GAG 1D4 NC-NC was insignificantly enhanced and lacked the ability to capture the additional p17p24. This suggests the steric hindrance introduced after interacting with the immobilized p24, causing improper orientation of the Ank GAG 1D4 NC-NC that limits the access of p17p24 to the second binding site. This data consensus with the MD simulation in which the binding surfaces of Ank GAG 1D4 NC-CN synchronically interact with CAp24. Half of the Ank GAG 1D4 NC-NC conformations preferred a 1:1 interaction, whereas most Ank GAG 1D4 NC-CN favored a 1:2 interaction. This phenomenon confers the potential of Ank GAG 1D4 NC-CN to interfere with HIV-1 Gag-assemble compared to Ank GAG 1D4 NC-NC and monomeric Ank GAG 1D4. Previously, an anti-HIV-1 production of Ank GAG 1D4-S45Y has been expressed, and the leakage of HIV-1 virions production was detected in the late infection [7]. Although Ank GAG 1D4-S45Y enhanced anti-HIV-1 activity, both dimeric ankyrins were superior. Additionally, the anti-HIV-1 activity of dimeric ankyrin was also investigated in the HIV-1 NL4-3 MIR CAI201V virus, which served as a CA-mutation virus model. Since this mutation occurs in CA-CTD, the monomeric and dimeric forms of Ank GAG 1D4 were presumed to inhibit HIV-1 NL4-3 MIR CAI201V virus replication. In this study, Ank GAG 1D4-S45Y and dimeric ankyrins showed a remarkable reduction in the viral progeny production in HIV-1 NL4-3 MIR CAI201V infection. This finding suggested that Ank GAG 1D4 NC-NC and Ank GAG 1D4 NC-CN retain their functionality to accommodate different CAp24 molecules promising to interfere with Gag packaging simultaneously. Interestingly, the bifunctional property of Ank GAG 1D4 NC-NC was not indicated by BLI, whereas the inhibitory effect was significantly improved in comparison with Ank GAG 1D4-S45Y. We hypothesized that the intercellular flexibility of Ank GAG 1D4 NC-NC is more compromised than in vitro. In addition, the dimension of Ank GAG 1D4 NC-NC is doubling of Ank GAG 1D4-S45Y, thus more efficient in perturbing capsid assembly. Further exploration should be performed to explain this phenomenon.
The results of this study suggested that the inverted second module of Ank GAG 1D4 NC-CN efficiently conforms to bifunctional activity. Considering the conformational structure of Ank GAG 1D4 NC-CN , it conducts a superior inhibitory response than Ank GAG 1D4 NC-NC . To provide the occupied dimension for CAp24, it is crucial to consider the effect of the extended distance between the binding surfaces apart from the flexibility of the linker. When dimeric ankyrins are expressed in the infected cells, they enhance the inhibitory activity against the HIV-1 NL4-3 WT virus. On days 7 and 13, the difference of CAp24 in the presence of Ank GAG 1D4-S45Y and dimers is significant compared to no ankyrin control (>1000-fold). However, after day 19 the control groups were terminated since the cell viability drastically decreased. Thus, on day 21 the CAp24 level was determined only with Ank GAG 1D4-S45Y and dimers. Most infected cells harboring Ank GAG 1D4-S45Y and dimers survived and continuously generated a small number of viral particles. Although the fold difference of CAp24 among this group is not much, the protection of dimers was significantly different from Ank GAG 1D4-S45Y in the WT virus. Moreover, dimeric ankyrins retain their viral protection activity against HIV-1 NL4-3 MIR CAI201V virus replication in SupT1 cells. Other drug-resistant strains regarding mutation at the capsid domain will be further evaluated. A substantial increase in pretreatment drug resistance prevalence indicated that first-line ART could not completely eradicate HIV-1 infection [25]. Based on our findings, gene therapy using Ank GAG 1D4 NC-CN could be feasible to integrate with highly active antiretroviral therapy (HAART) for sustainable treatment.
Dimeric ankyrin Ank GAG 1D4 NC-CN provides the avidity function regarding the flexible structure and the simultaneous accessibility of the interacting modules. The advantage of Ank GAG 1D4 NC-CN is the forceful interaction against the juxtaposed Gag molecules, thus efficiently interfering with the HIV-1 assembly process.

Construction of Inverted Ank GAG 1D4
The amino acid sequence of the inverted Ank GAG 1D4 is a backwardly read protein sequence of Ank GAG 1D4 (PDB ID: 4HLL) [26], which is used for the initial coordinates of the inversion. In the PDB file format, the atom serial number of the parent coordinates was rearranged in a mirror image order. The backbone atom names were subsequently modified. Two oxygens of the carboxyl group were removed, and the carbon atom of this group was swapped with the nitrogen atom of the amino group. The modified coordinate structure was eventually used as a template for the three-dimensional structure of the inverted Ank GAG 1D4. The structural model was constructed using the user-template mode of SWISS-MODEL software [27].

Structural Modeling of Ank GAG 1D4 Dimer
The initial three-dimensional model for the dimers was generated from the first ankyrin module 1D4 and connected to a flexible linker, Ank GAG 1D4-linker. The (G 4 S) 4 linker was extracted from the crystal structure of scFv-IL-1B (PDB ID: 2KH2) [28]. It was inserted with residues LLQ at the N-terminus and with residues TSDL at the C-terminus. The residues LLQ and TSDL were superimposed with the N-terminal and C-terminal amino acids of the first and second ankyrin modules, respectively. After superimposing the LL residues of Ank GAG 1D4 with the LL residues of the linker, two possible conformations of the Ank GAG 1D4-linker were generated. Atomistic molecular dynamics simulations of each Ank GAG 1D4-linker were performed using NAMD software version 1.1422 for 5 ns under the CHARMM36 force field [29]. TIP3P59 water was employed at 310 K and 1 atm. The folding dynamics were simulated with an integration time step of 2 fs, evaluated nonbonded every 1 fs, and updated electrostatics every 2 fs. Afterward, the last four residues (TSDL) of the MD Ank GAG 1D4-linker were superimposed with another Ank GAG 1D4 or an inverted Ank GAG 1D4 module to create the dimeric Ank GAG 1D4 NC-NC and Ank GAG 1D4 NC-CN , respectively. The dimers depicting the crashing structures of the ankyrin modules and linker were excluded. The remaining generated structures were analyzed for dimer behavior in interacting with CAp24. The duplex ankyrin modules were conclusively superimposed with the docking complex of Ank GAG 1D4 and CAp24 from a previous study [19]. The binding ratio of each dimer to CAp24 was also investigated.

Determination of the Distance of Binding Sites
The distance between the binding sites was measured between two carbon alpha atoms at L53 of the first and second modules of the Ank GAG 1D4 NC-NC dimer. The Ank GAG 1D4 NC-CN binding distance was determined from L53 of the first module to the reversed L53 residue in the second inverted module. The L53 of Ank GAG 1D4 was in the middle of S45 and Y56, which was used to confirm critical residues interacting with CAp24. The distance was calculated using NAMD software [30].

Analysis of the Binding Ratio of Ankyrin Dimer to CAp24
The geometry of CAp24 molecules extracted from the generated structures in the 'structural modeling of Ank GAG 1D4 dimer' described above was deployed to obtain the van der Waal (vdw) interactive energy using NAMD software. Suppose the vdw energy was ≤0, the 1:2 interaction without colliding with each captured CAp24 in binding with at 2500× g for 1.30 h and cultured for 16 h. After incubation, these cells were washed and cultured in a 10% HI-FBS-RPMI 1640 medium. Ankyrin-EGFP expression in cells was observed under an inverted fluorescence microscope. The percentage of EGFP-positive cells was determined by BD Accuri TM C6 (BD biosciences, Franklin Lakes, NJ, USA). SupT1 stable cells were sorted by a BD FACSMelody TM cell sorter (BD Biosciences, Franklin Lakes, NJ, USA) to obtain the comparable expression level of ankyrin.

Ankyrin Subcellular Localization
HeLa cells were transduced with VSV-G pseudotyped lentiviral vector at an MOI of 1, with 8 ug/mL of polybrene. VSV-G pseudotyped lentiviral vector carries ankyrins gene as mentioned above. After 48 h post-transduction, transduced cells were seeded on a glass coverslip overnight. Cells were incubated with Hoechst 33342 (ThermoFisher Scientific, Waltham, MA, USA) for nuclear staining. After washing, cells were fixed in 4% paraformaldehyde and mounted on a glass slide. Ankyrin localization was determined under fluorescent microscopy with apotome (100× magnification) using Zeiss Colibri 7. The mean fluorescent intensity was measured by ImageJ software (NIH). To compare the level of ankyrin expression at the plasma membrane, EGFP intensity, and ring area in ankyrin-expressing HeLa cells were calculated to intensity per area as follows: (outer ring intensity-inner ring intensity)/(outer ring area-inner ring area).

HIV-1 Infection
SupT1 cells (no ankyrin) and ankyrin-expressing SupT1 cells were inoculated with 10 MOI of HIV-1 NL4-3 WT or HIV-1 NL4-3 MIR CAI201V virus. In this experiment, SupT1 and Ank GAG 2D3-expressing SupT1 cells served as no protection control. After 16 h incubation, these cells were washed 3 times. Cells were cultured in RPMI 1640 medium supplemented with 10% HI-FBS and subcultured every 2 days. During culture, cell morphology was observed under an inverted microscope. Additionally, cell viability was monitored using CCK-8 assay. Briefly, 100 µL of cells were added to a 96-well plate and incubated with 10 µL of CCK-8 solution (Abbkine, Wuhan, China). Following 30 min incubation, viable cells were monitored at 450 nm by a CLARIOstar microplate reader (BMG Labtech, Baden-Württemberg, Germany).
To determine viral release in infected cells, culture supernatants at 7, 13, and 21 days post-infection were harvested for ELISA and real-time RT-qPCR as described below.

Viral Release Monitoring
The concentration of HIV-1 CAp24 in the culture supernatant was evaluated using Genscreen TM Ultra HIV-1 p24 ELISA kit (ELISA) (Bio-Rad, FR). The harvested culture supernatant was centrifuged to remove cell debris and unwanted particles. The viral particles in the culture supernatant were lysed with 1% Triton-X 100 before the assay. The absorbances at 450 nm were read using a microplate reader and calculated for HIV-1 CAp24 levels using the HIV-1 CAp24 standard curve. Additionally, HIV-1 production was monitored by quantifying HIV-1 RNA copy number in day 21 post-infection culture supernatant by real-time RT-qPCR using COBAS Ampliprep/COBAS TaqMan HIV-1 test (Roche, Basel, Switzerland).

Statistical Analysis
Data are presented as the mean ± SD from triplicate experiments. Statistical analysis was performed using one-way ANOVA. Statistical differences were considered significant at p ≤ 0.05 (indicated with asterisks).