Rafael Ceña Diez [1], Aswathy Narayanan [1], Maarten van de Klundert [1], Jimmy E Rodriguez [3], Kamal Singh [2], Ákos Végvári [3], Robert van Domselaar [1], Anders Sönnerborg [1,4]

Affiliates

1. Division of Infectious Diseases, ANA Futura Laboratory, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden 2. Bond Life Sciences Center, University of Missouri, Columbia, MO, USA. 3. Division of Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden 4. Division of Clinical Microbiology, ANA Futura Laboratory, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.

Abstract

Background: Understanding the natural control mechanisms in Elite controllers (Ecs) could contribute to the identification of new unique therapeutic strategies for functional HIV-1 cure. Previously, we reported the enrichment of certain dipeptides in blood plasma and feces of Ecs compared to therapy-naïve HIV-1 progressors, and HIV-1 negative controls. Among these dipeptides, tryptophylglycine (WG-am) showed the highest anti-viral activity against HIV-1 in vitro (Cena-Diez et al., 2022). The purpose of this study was to understand the mechanism by which WG-am is able to inhibit HIV-1.
Methods: Time of addition assay and other drug susceptibility assay (DSA) in PBMCs and TZM.bl were performed in other to elucidate the WG-am mechanism of action. A proteomic analysis of WG-am effect against HIV-1 was performed. Targeted RT-PCR to elucidate the antiviral mechanism.
Results: Molecular modelling indicated that WG-am was acting as attachment inhibitor by binding to gp120, thereby inhibiting the entry step. However, a time course assay showed that WG-am can also halt HIV-1 replication when added up to 6 hours post-infection, suggesting a second effect after the entry. DSA under acidic wash conditions did show the ability of WG-am to internalize into the host cell in an HIV independent manner. Proteomic analysis showed clustering of all samples treated with WG-am without regard of the number of doses or presence or absence of HIV-1. Proteomic data and DSA suggested that WG-am might have an effect on HIV-1 reverse transcription, which was later confirmed by real-time PCR.
Conclusions: Our data suggest that WG-am has two different mechanisms by which it inhibits HIV-1 replication. First, WG-am is an attachment inhibitor by interaction with the CD4 binding pocket of gp120. Secondly, WG-am exerts a post-entry antiretroviral effect through inhibition of the reverse transcription. Whether this effect is a direct or an indirect effect on the HIV-1 RT is still under investigation. The understanding of how WG-am contributes to the control of HIV-1 in ECs could add valuable information in the search for a functional cure of HIV-1 infection.