Performance of Affinity-Improved DARPin Targeting HIV Capsid Domain in Interference of Viral Progeny Production

Previously, a designed ankyrin repeat protein, AnkGAG1D4, was generated for intracellular targeting of the HIV-1 capsid domain. The efficiency was satisfactory in interfering with the HIV assembly process. Consequently, improved AnkGAG1D4 binding affinity was introduced by substituting tyrosine (Y) for serine (S) at position 45. However, the intracellular anti-HIV-1 activity of AnkGAG1D4-S45Y has not yet been validated. In this study, the performance of AnkGAG1D4 and AnkGAG1D4-S45Y in inhibiting wild-type HIV-1 and HIV-1 maturation inhibitor-resistant replication in SupT1 cells was evaluated. HIV-1 p24 and viral load assays were used to verify the biological activity of AnkGAG1D4 and AnkGAG1D4-S45Y as assembly inhibitors. In addition, retardation of syncytium formation in infected SupT1 cells was observed. Of note, the defense mechanism of both ankyrins did not induce the mutation of target amino acids in the capsid domain. The present data show that the potency of AnkGAG1D4-S45Y was superior to AnkGAG1D4 in interrupting either HIV-1 wild-type or the HIV maturation inhibitor-resistant strain.


Introduction
Human immunodeficiency virus (HIV) infection remains a major health problem worldwide. Highly active antiretroviral therapy (HAART) is currently used to sustain HIV suppression and recover the immune function of patients [1,2]. Despite success in terms of improved clinical symptoms, adverse drug effects from using HAART have been reported. Therefore, alternative strategies have been developed for HIV therapy [3]. Several intrabodies have been designed to target the viral HIV-1 protein, e.g., scFvD8 [4], GPI scFv-X5 [5], and scFv 183-H12-5C [6], which were generated to inhibit HIV-1 replication in infected cells. However, cytoplasmic reducing conditions halted their development, since proper folding and stability requires disulfide bond formation.
Accordingly, the attempt to construct a disulfide bond-independent scaffold might be promising for HIV-1 therapy. An alpha repeat (αRep) protein has been designed to target HIV-1 Gag. This αRep exhibits activity by impairing the viral packaging and maturation process [7,8]. Another type of disulfide bond-free scaffold is called designed ankyrin repeat protein (DARPin), which is based on natural ankyrin [9]. This building block provides the properties of ankyrin in protein-protein interactions involved in several cellular activities [10][11][12]. The advantages of DARPin include high stability and solubility. Furthermore, resistance in the protease and reducing cytoplasmic environment may make ankyrin an intracellular therapeutic molecule [10]. According to these advantages, DARPins were designed to overcome several limitations when using immunoglobulins as therapeutics agents [13][14][15][16]. In addition, the DARPins have been reported to have a role in HIV inhibition. CD4-specific DARPins [17] and HIV gp120-specific DARPins [18] were designed to block HIV-1 entry. However, their efficiency was reduced by unwanted side effects [19] and mutation in the HIV envelope [20].
Besides the extracellular anti-HIV-1 DARPins, we reported an intracellular anti-HIV-1 DARPin, Ank GAG 1D4, which specifically targets the N-terminus of the HIV-1 capsid protein [21]. Ank GAG 1D4 provides anti-HIV-1 activity through interference with HIV Gag multimerization, an important step in HIV assembly. This ankyrin reduces the permissiveness of HIV-1 production in HIV-1-infected SupT1 cells [22]. In addition, Ank GAG 1D4 has broad-spectrum antiviral activity against an HIV-1 circulatory strain that carries a mutation in the N-terminus capsid [23]. However, the anti-HIV-1 activity of Ank GAG 1D4 was mediocre, especially in the late stage of infection [24]. Computational analysis and calculation of van der Waals (vdW) forces indicate the choices of key amino acid residues in ankyrin sequence [25]. An evaluation of the binding activity and affinity using an enzyme linked immunosorbent assay (ELISA)-modified method and bio-layer interferometry (BLI) showed that substitution of serine (S) at position 45 with tyrosine (Y), forming Ank GAG 1D4-S45Y, leads to increased affinity against the HIV-1 capsid domain. Enhanced binding affinity of Ank GAG 1D4 might provide complete HIV-1 inhibition.
The emergence of drug-resistant strains is another important obstacle in HIV-1 therapy. Mutations in the genes involved with antiretroviral drug target sites are continuously reported [26,27], resulting in the failure of HAART. Nowadays, several HIV-1 drugs and inhibitors have been developed in order to overcome this problem [28]. Capsid-targeting inhibitors represent one interesting compound, which work by interfering in the late stage of the HIV-1 life cycle, assembly and maturation [29,30]. The HIV-1 maturation inhibitor (MI) is a class of anti-HIV-1 compound that blocks proteolytic cleavage of the Gag protein, resulting in non-infectious virions. MI can be divided into two classes; betulinic acidbased and pyridone-based MI. The betulinic acid-based MI, bevirimat (BVM), blocks HIV-1 maturation by interrupting CA-SP1 cleavage [31]. According to the resistance-conferring mutation on the Gag protein, a BVM derivative, C28-BVM, was further developed [32]. The second class of MI, PF46396, exhibits antiretroviral activity in HIV-1 laboratory strain and HIV-1 circulatory isolates. However, HIV-1 resistance against both classes of MI has been reported [33][34][35]. These data indicate that even though new anti-HIV-1 agents were developed, it is not enough to inhibit HIV-1 replication. As the target region of Ank GAG 1D4 is distinctive from that of MI, Ank GAG 1D4 is expected to inhibit the assembly process of the HIV-1 MI-resistant strain.
This study was aimed at investigating the anti-HIV activity of binding affinityenhanced Ank GAG 1D4 in infected SupT1 cells. In addition, the role of the Ank GAG 1D4 in HIV-1 maturation inhibitor resistant (MIR) strain was addressed. The HIV-1 NL4-3 MIR CAI201V virus, carrying a mutation on the CA-SP1 junction on Gag protein, was used as a model. Regarding our results, the binding affinity-improved Ank GAG 1D4 had increased antiviral activity against wild-type (WT) and MIR viruses.
pNL4-3 plasmid, the infectious HIV-1 NL4-3 molecular clone (NIH), was used to produce the HIV-1 NL4-3 laboratory strain virus. Additionally, mutagenesis was performed on this plasmid to generate a clone of the HIV-1 MIR virus.

Construction of pNL4-3 MIR CAI201V Plasmid and Preparation of HIV-1 Maturation Inhibitor Resistant (MIR) Virus
In order to compare the function of Ank GAG 1D4 and Ank GAG 1D4-S45Y in HIV-1 MIR virus production, HIV-1 NL4-3 MIR CAI201V was generated. Mutation at position 201 on CA-CTD from isoleucine (I) to valine (V), CAI201V, confers resistance of HIV-1, in both clade B and C, against PF-46396, and partial resistance to BVM [33,35]. To construct the molecular clone of HIV-1, NL4-3 MIR CAI201V , mutagenesis was performed on pNL4-3 plasmids using a QuickChange Lightening Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). The synthesized oligonucleotides used in this experiment were as follows: Fwd_CAI201V: 5 cgaacccagattgtaagactgtgttaaaagcattgggacca-3 ; Rev_CAI201V: 5 tggtcccaatgcttttaacacagtcttacaatctgggttcg-3 . The mutated plasmid was transformed into XL-1 blue competent E. coli cells for plasmid amplification. The plasmid-harboring XL-1 blue cells were grown on Luria-Bertani (LB) agar supplemented with 100 µg/mL of ampicillin, at 37 • C for 16 h. A bacterial colony was picked and further cultured in super broth (SB) supplemented with 100 µg/mL of ampicillin at 37 • C for 16 h. After culturing, the plasmid was extracted and purified using a Geneaid™ Midi Plasmid Kit (Geneaid Biotech, New Taipei, Taiwan). To confirm the corrected mutagenesis, pNL4-3 MIR CAI201V was subjected to plasmid sequencing analysis.
pNL4-3 MIR CAI201V was used for HIV-1 NL4-3 MIR CAI201V viral production. This plasmid was transfected into HEK293T cells using MirusTransITX2 (Mirus Bio, Madison, WI, USA). After 48 h post-transfection, culture supernatant containing virus was harvested, centrifuged at 335× g for 5 min, and filtrated through 0.45 µm filter membrane to remove unwanted particles. The viral stock was aliquoted and kept at −80 • C. HIV-1 viral titer was determined by HIV viral load assay using COBAS Ampliprep/COBAS Taqman HIV-1 test (Roche, Basel, Switzerland). To generate SupT1 cells stably expressing ankyrin protein, 1 × 10 5 of SupT1 cells were transduced with VSV-G pseudotyped lentiviral vector at a multiplicity of infection (MOI) of 1, with the addition of 5 ug/mL polybrene. Each VSV-G pseudotyped lentiviral vector included VSVG-CGW-Myr (+) Ank GAG 1D4-EGFP, VSVG-CGW-Myr (+) Ank GAG 1D4-S45Y-EGFP, and VSVG-CGW-Myr (+) Ank A3 2D3-EGFP. These cells were spinoculated at 2500× g for 1.30 h, and further cultured for 16 h. After incubation, these cells were washed 3 times with RPMI 1640 medium and cultured in 10% heat-inactivated FBS-RPMI 1640. To evaluate ankyrin expression in SupT1 cells, EGFP-positive cells were observed under an inverted fluorescence microscope (Zeiss Axio Observer-Colibri 7) and the percentage of EGFP-positive cells was determined by a CyAn TM ADP flow cytometer (Beckman Coulter, Brea, CA, 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. 2.6. Evaluation of CD4 surface expression on SupT1 was done on cells stably expressing ankyrin protein. To test whether overexpression of ankyrin in SupT1 cells interferes with CD4 expression on the cell surface, CD4 protein was examined by immunofluorescence staining. SupT1 cells and SupT1 stable cells were washed twice with phosphate-buffered saline (PBS) and incubated in 10% human AB serum-PBS on ice for 30 min. After incubation, the cells were stained with APC-conjugated mouse anti-human CD4 antibody (Immunotools, Friesoythe, Germany) and placed on ice for 30 min. Next, cells were washed 3 times with FACS buffer solution and resuspended in fixation buffer (1% paraformaldehyde in PBS). CD4-positive cells were analyzed by a BD Accuri TM C6 cytometer (BD biosciences, Franklin Lakes, NJ, USA).

Determination of Subcellular Localization of Ankyrin Proteins in SupT1 Cells
To determine subcellular localization of ankyrin proteins, ankyrin-EGFP-expressing SupT1 cells were observed under confocal fluorescence microscopy. SupT1 cells and ankyrin-EGFP-expressing SupT1 cells were centrifuged at 335× g for 5 min, then resuspended in RPMI 1640 medium. A total of 1 × 10 6 cells of SupT1 cells or ankyrin-EGFPexpressing SupT1 cells were seeded to poly-L lysine-precoated cover slips. Cells were incubated in humidified a 5% CO 2 atmosphere incubator at 37 • C for 10 min. Cells were subsequently incubated in fixation buffer (4% paraformaldehyde in PBS) at room temperature for 15 min. After twice washing with PBS, cells were stained with a 1:1000 dilution of CellMask TM Deep red membrane staining (Thermo Fisher Scientific, Waltham, MA, USA) and 1:1000 dilution of Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) in RPMI 1640 medium at 37 • C for 10 min. Cell imaging was performed using Nikon C2 plus confocal fluorescence microscopy (Nikon, TYO, Japan) at 63× magnification. Excitation wavelengths were 405 nm for Hoechst 33342, 488 nm for EGFP, and 560 nm for CellMask TM Deep red membrane staining.

Evaluation of HIV-1 p24 and Viral Load
The level of HIV-1 capsid (p24) in culture supernatant was evaluated using a Genscreen TM Ultra p24 ELISA kit (Bio-Rad, Marnes-la-Conquette, PAR, France). The viral particles in culture supernatant were lysed by 1% Triton-X 100 prior to the assay. Culture supernatants were added to a well precoated with monoclonal antibody against HIV-1 p24. After incubation and washing, biotinylated anti-HIV-1 p24 polyclonal antibody was added. The reaction was incubated at room temperature for 30 min, then washed. Next, HRP-conjugated streptavidin was added to the well, and the reaction was incubated for 30 min at room temperature. After incubation and washing, the reaction was detected by adding chromogenic substrate, and stopped at 30 min with 1 N sulfuric acid solution. The absorbances were read using a microplate reader at 450 nm, and calculated for HIV-1 p24 levels using HIV-1 p24 standard curve. To further determine the viral production, culture supernatants at 13 days post-infection were subjected to HIV viral load assay. The level of HIV virion in culture supernatant was evaluated using reverse transcription quantification polymerase chain reaction (RT-qPCR) by COBAS Ampliprep/COBAS Taqman HIV-1 test (Roche, Basel, Switzerland).

Analysis of HIV-1 Capsid Sequence
The sequence analysis of the HIV-1 capsid was modified from a previously described method [22]. In brief, viral RNA was extracted from culture supernatant using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). To generate cDNA encoding the HIV-1 capsid, extracted RNA was used as a template for reverse transcription PCR (RT-PCR) using a Superscript III One-step RT-PCR system (Invitrogen, Friesoythe, Germany). The PCR reaction contained a pair of oligonucleotides, (FWD_RIHES_p24: 5 -ggatagaggtaaaagacaccaaggaagc-3 ; REV_RIHES_p24: 5 -ctcattgcctcagccaaaacccttgc-3 ), and PCR product was purified using a GENEJET PCR purification kit (Thermo Fisher Scientific, Waltham, MA, USA). The purified PCR product was subjected to DNA sequencing with the same oligonucleotides with RT-PCR. To analyze sequencing results, HIV-1 capsid sequence was aligned against HIV-1 NL4-3 WT using SnapGene software version 2.8.3 (GSL Biotech, San Diego, CA, USA).

Statistical Analysis
The data are presented as the mean ± S.D. from 3 replicate experiments. Statistical analysis was performed using unpaired t-test. Differences were considered significant at p < 0.05 (indicated with asterisks).

Expression of Ankyrin Protein Did Not Interfere with Cell-Surface CD4
To generate SupT1 stable cells, SupT1 cells were transduced with VSV-G pseudotyped lentivirus. Each lentivirus vector carries the gene encoding N-terminus myristoylated ankyrin protein with enhanced green fluorescence protein (EGFP) fusion, including Myr (+) Ank A3 2D3-EGFP, Myr (+) Ank GAG 1D4-EGFP, and Myr (+) Ank GAG 1D4-S45Y-EGFP. After 48 h post-transduction, SupT1 cells were observed to be EGFP positive under fluorescence microscopy ( Figure 1A). Since a comparable expression level of ankyrin is required to verify their anti-HIV-1 activity in infected cells, these transduced SupT1 cells were sorted. After cell sorting, the percentage of EGFP-positive cells in SupT1 stable cells was approximately 100%, with a comparable level of ankyrin ( Figure 1B,C). In addition, subcellular localization of ankyrin proteins in SupT1 cells was determined under confocal microscopy. The result showed that EGFP tagging ankyrins were colocalized with a plasma membrane dye, suggesting their targeting to inner leaflet of cell membrane as a result from the N-terminus myristoylation signal (Figure 2). To examine whether the expression of ankyrin interfered with the HIV receptor, CD4 expression on the surface of SupT1 stable cells was measured by flow cytometry. The results demonstrated that the number of CD4-positive cells in ankyrin-expressing and control SupT1 cells was similar ( Figure 3A). The mean fluorescence intensity of CD4 in SupT1 cells and ankyrin-expressing SupT1 cells (Myr (+) Ank A3 2D3-EGFP, Myr (+) Ank GAG 1D4-EGFP, and Myr (+) Ank GAG 1D4-S45Y-EGFP) was 4.47 × 10 4 , 5.58 × 10 4 , 4.31 × 10 4 , and 3.96 × 10 4 , respectively ( Figure 3B). These data suggest that the expression of N-terminus myristoylated ankyrin protein did not alter the level of CD4 on the cell surface of SupT1 cells.

Anti-HIV-1 Ankyrins Do Not Drive Mutation in Amino Acid Sequence of HIV-1 Capsid
According to the infection experiment, leakage of viral progeny was detected on the last day of observations. Therefore, we determined whether the leakage in protection was a result from mutation in the ankyrin-targeted region. Since our anti-HIV-1 ankyrins were against the HIV-1 capsid, viral cDNA was subjected to sequencing for capsid amino acid sequence analysis. According to the alignment result, no mutation was indicated, especially in helix 1 and helix 7 (Figure 7), targeting regions of ankyrin on the N-terminus capsid. These data suggest that the leakage of HIV-1 progeny was due to an overload of virus. In addition, Ank GAG 1D4 and Ank GAG 1D4-S45Y did not drive mutation in the HIV-1 capsid.

Binding Affinity-Enhanced Ankyrin Provides Antiviral Effects on HIV-1 Maturation Inhibitor Resistant Virus
To solve the drug resistance issue, several anti-HIV-1 compounds were established; the HIV-1 maturation inhibitor is one anti-HIV-1 compound. Although these anti-HIV-1 compounds performed well in inhibiting HIV-1 production, a number of MI-resistant strains were reported. In this study, the antiviral activity of ankyrin on HIV-1 MIR virus was investigated. HIV-1 NL4-3 MIRCAI201V was selected as a model to observe intracellular anti-HIV-1 activity of ankyrin. SupT1 cells and ankyrin-expressing SupT1 cells were infected with HIV-1 NL4-3 MIRCAI201V virus at 10 MOI. After HIV-1 challenge, the infected cells were observed for syncytium formation under microscopy ( Figure S5). Infected SupT1 cells and SupT1/Myr (+) Ank A3 2D3 cells showed no protection against HIV-1 replication. A number of syncytial cells were observed on day 13 in SupT1 cells and SupT1/Myr (+) Ank A3 2D3 cells with the appearance of clumping cells ( Figure 8A). Conse-

Binding Affinity-Enhanced Ankyrin Provides Antiviral Effects on HIV-1 Maturation Inhibitor Resistant Virus
To solve the drug resistance issue, several anti-HIV-1 compounds were established; the HIV-1 maturation inhibitor is one anti-HIV-1 compound. Although these anti-HIV-1 compounds performed well in inhibiting HIV-1 production, a number of MI-resistant strains were reported. In this study, the antiviral activity of ankyrin on HIV-1 MIR virus was investigated. HIV-1 NL4-3 MIR CAI201V was selected as a model to observe intracellular anti-HIV-1 activity of ankyrin. SupT1 cells and ankyrin-expressing SupT1 cells were infected with HIV-1 NL4-3 MIR CAI201V virus at 10 MOI. After HIV-1 challenge, the infected cells were observed for syncytium formation under microscopy ( Figure S5). Infected SupT1 cells and SupT1/Myr (+) Ank A3 2D3 cells showed no protection against HIV-1 replication.

Discussion
Although HAART is successfully used for HIV-1 therapy, it is limited by adverse drug effects and viral mutation. Furthermore, development of HIV-1 drugs takes years and is expensive [37]. It is desirable to establish new anti-HIV molecules against alternative viral targets in the HIV life cycle. Instead of antibodies and their derivatives, DARPins, representing a disulfide-independent scaffold, were sought for HIV-1 therapy based on their biological properties [3,38,39]. An extracellular DARPin, including CD4-specific DARPins and gp120-specific DARPins, was reported to inhibit HIV-1 entry [17,18]. Although these DARPins specifically perceive their target, limitations in terms of immune function and mutation-driven side effects were reported. CD4-specific DARPins can lead to impaired CD4 function, while gp120-specific DARPins drive mutation in the HIV-1 envelope. Moreover, a high clearance rate of DARPins in the blood circulation remains an obstacle [19]. Accordingly, we previously generated an intracellular Ank GAG 1D4, which interferes with HIV-1 assembly by interacting with the N-terminus of HIV-1 capsid domain (CA-NTD) [21]. Several studies indicated that this specific area is crucial in viral assembly, maturation, and uncoating through viral capsid mutation [40,41]. The mutation leads to capsid polymorphisms that impair HIV-1 infectivity. Numerous CA-targeted molecules have been studied, such as PF74 [42], CAI [43], and GSCAI [16]. Although these molecules express activity in inhibiting HIV-1 replication, viral escape and inefficient cell penetration hamper its competency [44]. In contrast, DARPin is well-expressed inside the cells [45], specifically Ank GAG 1D4, with the domain necessary for capsid polymorphism [21].
Despite the demonstrated anti-HIV-1 activity of Ank GAG 1D4, protection in the late period of in vitro culture needs to be improved [24]. To enhance the efficiency of Ank GAG 1D4, computational analysis was performed to identify the key residues on the ankyrin binding sites suitable for mutagenesis [25]. According to previous work, substituting tyrosine for serine improves the binding affinity of Myr (+) Ank GAG 1D4-S45Y without altering specificity. However, an investigation of the intracellular anti-HIV activity of affinity-enhanced Ank GAG 1D4 is required. In this study, antiviral activity of Myr (+) Ank GAG 1D4-S45Y was observed in infected SupT1 cells compared with parental Myr (+) Ank GAG 1D4. The affinityimproved ankyrin expression delayed the formation of syncytium cells and dramatically decreased viral replication as measured by p24 and viral load. By and large, different from mutant ankyrin, HIV-1 Gag was impaired in the assembly process, leading to reduced HIV-1 production in infected cells. The cytopathic effect in HIV-1 infected cells in the form of multinucleated giant cell formation and cell rupture represents a mechanism involving HIV-1 replication [46]. According to our experiment, inhibition of HIV-1 replication by Ank GAG 1D4-S45Y results in late detection of syncytium formation, followed by extended cell viability. Although minimal progeny leakage was evidenced on day 21, the interactive amino acid sequence at HIV-1 NL4-3 CA was conserved. This suggests that both ankyrins were not likely to drive the mutation of HIV-1 NL4-3 CA. Thus, the progeny detected on the last day of culture probably resulted from the overload of HIV particles, which exceeded the ankyrin harness.
From our results, tyrosine substitution introduces binding affinity to intracellular Gag, since tyrosine is frequently found in the hot-spot of the antigen binding site [47]. Additionally, the role of tyrosine in mediating the binding of Ank GAG 1D4 against a viral target has been highlighted by computational analysis and in vitro studies [25]. Tyrosine contains an aromatic side chain, comprising both a hydrophobic ring and a hydrophilic hydroxyl group, which contributes to its hydrogen bond forming ability, hydrophobic interaction, van der Waals interaction, and amino aromatic interaction [48,49]. Replacement with tyrosine provides more stable interaction of Ank GAG 1D4-S45Y against viral targets, leading to higher efficiency in anti-viral activity.
Another group of CA-binding compounds is HIV-1 MIs. MIs confer anti-HIV activity through disrupting the maturation process of the virus at the CA-SP1 junction. However, HIV-1 is highly sensitive to mutations at the CA-SP1 junction, resulting in reports of MIresistant strains [50]. Different from MI, our anti-assembly ankyrin specifically interacted with CA-NTD [21]. We assumed that Ank GAG 1D4 inhibits replication of resistant viruses at the step prior to maturation. To prove the concept, we investigated the activity of Ank GAG 1D4 and its mutant against MI-resistant virus. SupT1 cells expressing Ank GAG 1D4 and Ank GAG 1D4-S45Y were infected with HIV-1 NL4-3 MIR CAI201V virus. This mutation has been reported to confer resistance against PF4696, the second-class HIV-1 maturation inhibitor, and partial resistance to BVM [33,34]. From the infection experiment, both anti-HIV-1 ankyrins were shown to have a negative effect on viral replication. Herein, HIV-1 NL4-3 MIR CAI201V virus is a representative for observing antiviral activity against drug-resistant viruses. Another mutation on the HIV-1 capsid and downstream of the SP1 region also confers MI resistance [51,52]. Moreover, there are several reports on mutations in the HIV-1 genome, leading to the emergence of HIV-1 drug resistance against first-line ART [27] as well as a novel HIV-1 drug classes. For example, the M184V mutation along with thymidine analogue-associated mutations (TAMs) in HIV-1 reverse transcriptase gene increases abacavir resistance [53]. Another is Gag cleavage site mutation, which may confer resistance against protease inhibitors (PIs) in patients who fail PI-containing regimens [54]. As a different viral target site, Ank GAG 1D4-S4Y might inhibit the viral replication of these resistant strains of HIV.

Conclusions
Our current results underscore the significance of Ank GAG 1D4-S45Y for enhancing antiviral activity in either WT HIV-1 NL4-3 or MIR virus. Although the single amino acid change in a previous report did not markedly increase the affinity of Ank GAG 1D4, the intracellular activity of Ank GAG 1D4-S45Y demonstrated distinctly notable performance. Further improvement of Ank GAG 1D4 affinity will provide a direction for rational design regarding predicted complexes from molecular dynamics (MD) simulations.