Broad-Spectrum Antiviral Activity of an Ankyrin Repeat Protein on Viral Assembly against Chimeric NL4-3 Viruses Carrying Gag/PR Derived from Circulating Strains among Northern Thai Patients

Abstract

Certain proteins have demonstrated proficient human immunodeficiency virus (HIV-1) life cycle disturbance. Recently, the ankyrin repeat protein targeting the HIV-1 capsid, Ank^GAG1D4, showed a negative effect on the viral assembly of the HIV-1_NL4-3 laboratory strain. To extend its potential for future clinical application, the activity of Ank^GAG1D4 in the inhibition of other HIV-1 circulating strains was evaluated. Chimeric NL4-3 viruses carrying patient-derived Gag/PR-coding regions were generated from 131 antiretroviral drug-naïve HIV-1 infected individuals in northern Thailand during 2001⁻2012. SupT1, a stable T-cell line expressing Ank^GAG1D4 and ankyrin non-binding control (Ank^A32D3), were challenged with these chimeric viruses. The p24CA sequences were analysed and classified using the K-means clustering method. Among all the classes of virus classified using the p24CA sequences, SupT1/Ank^GAG1D4 demonstrated significantly lower levels of p24CA than SupT1/Ank^A32D3, which was found to correlate with the syncytia formation. This result suggests that Ank^GAG1D4 can significantly interfere with the chimeric viruses derived from patients with different sequences of the p24CA domain. It supports the possibility of ankyrin-based therapy as a broad alternative therapeutic molecule for HIV-1 gene therapy in the future.

Keywords: Gag polyprotein; HIV-1; ankyrin; protein therapy; virus assembly inhibitor.

Figure 1

Establishment of SupT1 cells stably expressing Ank^GAG1D4 and Ank^A32D3 fused to an enhanced green fluorescent protein (EGFP) reporter protein. The EGFP reporter protein expression and the morphology of the cells were determined using an inverted fluorescence microscope at 200× magnification (top panel) at 48 h post-transduction. The percentages of EGFP-positive cells, which represent the SupT1 cells expressing Ank^GAG1D4 and Ank^A32D3, were investigated using flow cytometry (bottom panel).

Figure 2

The p24 capsid domain (p24CA) levels in the supernatant collected from SupT1 cells harbouring Ank^GAG1D4 and Ank^A32D3 after infection with 131 Gag/PR chimeric viruses. (A) Distribution of p24CA showed that the mean ± SEM of SupT1/Ank^A32D3 and SupT1/Ank^GAG1D4 was 186.5 ± 25.8 µg/mL and 62.2 ± 16.0 µg/mL, respectively. (B) Significant reduction of p24CA in SupT1/Ank^GAG1D4 compared with SupT1/Ank^A32D3 by individual samples. ** p < 0.01 was determined using a two-tailed paired Student t-test. The percentage of samples that experience reduction was 98.47%.

Figure 3

Quantitative analysis of the p24CA levels of the four clusters. (A) Four clusters, namely C1, C2, C3, and C4, were classified by the K-means clustering method. In the case of C4, a dataset in which the p24CA levels of SupT1/Ank^GAG1D4 and SupT1/Ank^A32D3 were over 1500 µg/mL was excluded from the plot. (B) The p24CA level of the four clusters after being classified by the K-means clustering method. Significant differences (** p < 0.01) in the p24CA levels between SupT1/Ank^GAG1D4 and SupT1/Ank^A32D3 were determined using the two-tailed Wilcoxon matched-pairs signed-rank test.

Figure 4

Ank^GAG1D4 inhibits syncytium formation of SupT1 cells after being infected by Gag/PR chimeric viruses induction. SupT1/Ank^GAG1D4 and SupT1/AnkG^A32D3 were infected with chimeric viruses. The cells were observed at 400× magnification using an inverted microscope. The black arrows point to syncytia. DK001 and DK007 are two representative samples of the Gag/PR chimeric viruses in cluster 1.

Figure 5

The consensus sequence and amino acid distribution of NTD^p24 in 131 Gag/PR chimeric viruses. For each NTD^p24 position, the HXB2 index is shown at the top, followed by the NTD^p24CA interface, corresponding consensus amino acid, and prevalence of particular residues. The NTD^p24CA–NTD^p24CA interface and the NTD^p24CA–CTD^p24CA interface (distance < 5 Å) are marked in green and yellow, respectively. The interface residues making the hydrogen bond are in dark green (NTD^p24CA–NTD^p24CA) and brown (NTD^p24CA–CTD^p24CA). The symbols in colour denote the Ank^GAG1D4 binding pocket residues. The bullets, diamonds, and boxes are the binding residues in the complexes of Ank^GAG1D4 bound to helix 1, those bound to helix 7, and those binding both helices, respectively. The corresponding consensus amino acid was defined by the most prevalent of the particular residues. Polymorphisms with proportions ≥5% are indicated using blue superscripts, and red otherwise. The amino acids in the blue box are alternative characters of the key residues interacting with Ank^GAG1D4, whereas the amino acids in the red box are variable residues.

Figure 6

Nineteen amino acids having polymorphic residues present in the docking complexes of Ank^GAG1D4 (PDB code: 4HLL and NTD^p24CA (PDB code: 2LF4). Ank^GAG1D4 bound to helix 1 (A) Ank^GAG1D4 bound to helix 7 (B). The atom style represents the residues on the NTD^p24CA (in ribbon style) interacting with Ank^GAG1D4 (surface form). The colour of the atom style indicates the distance between the NTD^p24CA residues and Ank^GAG1D4 in which pink, cyan, dark green, light green, yellow, and orange indicate distances at <5 Å, 5–10 Å, 10–20 Å, 20–30 Å, and 30–40 Å, >40 Å, respectively. The variable residue, S120 (40.46%), is outside the binding pocket residues in the case of Ank^GAG1D4 bound to helices 1 and 7 of the NTD.