The Effect of Cationic DNA-Binding Proteins on HIV-1 Latency.

Abstract

The latent reservoir remains the foremost obstacle to a HIV-1 cure development. This latent reservoir is composed of cells infected with replication-competent and yet transcriptionally silent proviruses, which are the source of viral rebound after antiretroviral therapy (ART) is interrupted. Therefore, people living with HIV-1 (PLWH) have to remain on treatment for their lifetime. However, ART is associated with cytotoxicity and comorbidities thus necessitating cure development. Despite extensive investigation, existing cure strategies, targeting HIV-1 latency such as the “shock and kill” approach, which uses latency-reversing agents (LRAs) to reactivate latent virus and expose infected cells to immune-mediated clearance, and the “block and lock” strategy, which employs latency-promoting agents (LPAs) to force the virus into deep latency, have shown limited long-term success. This underscores a need to continue the search for better and more effective agents for these HIV-1 cure strategies. Lysozyme and lactoferrin are cationic antimicrobial proteins that play an important role in innate defence and have both been shown to block HIV-1 replication, bind DNA and RNA, and modulate gene expression. However, the effect of these cationic proteins on HIV-1 latency potential remains to be determined. Therefore, the aim of this study is to determine the effect of cationic proteins of the innate immune system on the propensity of HIV-1 latency reversal or enhancement. The cytotoxicity of cationic proteins, chicken egg lysozyme (Lz), human lysozyme fragment (HL9), and/or lactoferrin (Lf), alone or in combination with established latency-reversing agents (LRAs; PMA and SAHA) and latency-promoting agents (LPAs; tanespimycin [Ts] and spironolactone [Sp]) was assessed in two HIV-1 latency cell lines, J-Lat A2 and J-Lat C, derived from a Jurkat E6 T-cell clone, using the Cell Counting Kit-8 assay. J-Lat A2 and J-Lat C cell lines harbour a latent minimal HIV-1-based retroviral vector reporter genome expressing subtype B (J-Lat A2) or subtype C (J-Lat C) consensus transactivator of transcription protein (Tat) together with green fluorescent protein (GFP), under the control of the corresponding subtype B or C consensus long terminal repeat (LTR), respectively. J-Lat A2 and J-Lat C were treated with individual cationic proteins Lz, HL9, or Lf, or in combination with PMA or SAHA and Ts or Sp. HIV-1 latency reactivation or enhancement was assessed after 24-48 hours by measuring GFP expression using flow cytometry. Cytotoxicity assays revealed that all agents were non-toxic (≥85% viability) at tested concentrations and combinations. Our data from single-treatment latency-reversal experiments showed that only Lf significantly reactivated latent HIV-1 in J-Lat A2 (3.6 ± 1.4 % GFP, p < 0.0009) but not in J-Lat C (1.4 ± 0.6 %, p = 0.997). Combinations Lz+Lf (4.9 ± 1.7 %, ≈25-fold, p < 0.0001) and Lz+HL9+Lf (6.6 ± 2.9 %, ≈34-fold, p < 0.0001) synergised and enhanced reactivation in J-Lat A2. Lactoferrin also enhanced classical LRAs, PMA-induced reactivation rose 2.3 ± 0.1-fold and SAHA 18.9 ± 2.3-fold (p < 0.0001) in J-Lat A2. The Lf+Lz combination raised these to 2.6- and 24.5-fold, respectively, versus LRA-only controls (p < 0.0001). The same pairs raised PMA 1.24 ± 0.2-fold (p = 0.02) and SAHA 9.5 ± 2.6-fold (p < 0.0001) in the J-Lat C cell line. The LPA Ts suppressed PMA-induced reactivation 9.0 ± 1.4-fold (p < 0.0001) in J-Lat A2. The inclusion of Lf restored the potent levels of reactivation, with combinations PMA+Ts+Lf and PMA+Ts+Lf+Lz resulting in 6.6 ± 0.5- and 7.8 ± 0.5-fold increases in GFP expression, respectively, relative to the PMA+Ts baseline (p < 0.0001). Although Ts did not block SAHA-induced reactivation, SAHA+Ts+Lf and SAHA+Ts+Lz+Lf combinations markedly potentiated SAHA, yielding 12.5 ± 1.4-fold and 15.5 ± 1.5-fold increases in GFP-expression compared to SAHA+Ts baseline (p < 0.0001). Data generated from the J-Lat C cell line followed a similar but less pronounced pattern where the combinations PMA+Ts+Lf, PMA+Ts+Lf+Lz, SAHA+Ts+Lf and SAHA+Ts+Lz+Lf significantly potentiated LRA-induced reactivation to levels that surpassed the LRA+LPA-only baseline. Sp reduced PMA-driven GFP expression 1.0 ± 0.07-fold compared to PMA alone (p < 0.0001); SAHA+Sp showed only marginal suppression. The Lz+Lf pair completely reversed Sp-induced suppression, PMA+Sp+Lz+Lf yielded 2.7 ± 0.7-fold and SAHA+Sp+Lz+Lf 26.4 ± 8-fold reactivation (both p < 0.0001). In J-Lat C, PMA+Sp+Lz+Lf (3.6-fold, p < 0.0001) and SAHA+Sp+Lz+Lf (2.2-fold, p = 0.0029) again overcame Sp-mediated deep latency. These findings suggest Lf, alone or in combination with Lz, can enhance latency reversal in permissive contexts and potentially counteracts LPA’s blocking effects, which was more pronounced in J-Lat A2 compared to J-Lat C. This highlights Lf as a promising agent that can be further explored for utility in the “shock and kill” cure strategy, and underscores the importance of cellular and viral subtype context in therapeutic outcomes.