Benzyl-2-Acetamido-2-Deoxy-α-d-Galactopyranoside Increases Human Immunodeficiency Virus Replication and Viral Outgrowth Efficacy In Vitro

Abstract

Glycosylation of host and viral proteins is an important posttranslational modification needed to ensure correct function of glycoproteins. For this reason, we asked whether inhibition of O-glycosylation during human immunodeficiency virus (HIV) in vitro replication could affect HIV infectivity and replication rates. We used benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (BAGN), a compound that has been widely used to inhibit O-glycosylation in several cell lines. Pretreatment and culture of PHA-blast target cells with BAGN increased the percentage of HIV-infected cells (7.6-fold, p = 0.0115), the per-cell amount of HIV p24 protein (1.3-fold, p = 0.2475), and the viral particles in culture supernatants (7.1-fold, p = 0.0029) compared to BAGN-free cultures. Initiating infection with virus previously grown in the presence of BAGN further increased percentage of infected cells (30-fold, p < 0.0001), intracellular p24 (1.5-fold, p = 0.0433), and secreted viral particles (74-fold, p < 0.0001). BAGN-treated target cells showed less CD25 and CCR5 expression, but increased HLA-DR surface expression, which positively correlated with the number of infected cells. Importantly, BAGN improved viral outgrowth kinetics in 66% of the samples tested, including samples from HIV controllers and subjects in whom no virus could be expanded in the absence of BAGN. Sequencing of the isolated virus indicated no skewing of viral quasi-species populations when compared to BAGN-free culture conditions. BAGN also increased virus production in the ACH2 latency model when used together with latency-reversing agents. Taken together, our results identify BAGN treatment as a simple strategy to improve viral outgrowth in vitro and may provide novel insights into host restriction mechanisms and O-glycosylation-related therapeutic targets for HIV control strategies.

Novel Approaches Towards a Functional Cure of HIV/AIDS

“…However, qVOA is time intensive and requires large amounts of biological specimen. In addition, qVOA may actually underestimate the size of the clinically relevant reservoir due to sampling limitations of latently infected reservoir cells from the most critical body compartments and the inability to reactivate all intact virus-carrying cells in vitro [20]. As an alternative to qVOA, different assays are now available to determine the inducible HIV reservoir and the viral transcriptional activity, including TILDA (Tat/Rev-induced limiting dilution assay) [16], IPDA (intact pro-viral DNA assay) and Quadruplex qPCR (Q4PCR, [18,21,22].…”

N-Glycosylation of TREK-1/hK2P2.1 Two-Pore-Domain Potassium (K2P) Channels

“…Finally, to assess whether hTREK-1 channel subunits might be subjected to O -glycosylation, plasmid DNA encoding WT and completely N -glycosylation-deficient N110Q/N134Q channel subunits was transiently transfected into HEK-293T cells. Subsequently, the cells were treated with the O -glycosylation inhibitor benzyl 2-acetamido-2-deoxy-α- d -galactopyranoside (BenGal), which acts as a mimic of GalNAc-α-1- O -serine/threonine and thus completely inhibits O -glycan chain extension by blocking the β-1,3-galactosyltransferase involved in elongation of O -glycosides [44,45,46]. In a subset of samples, protein lysates were subjected to digestion with O -glycosidase.…”

O‐glycosylation in viruses: A sweet tango

“…Moreover, it was discovered that the administration of benzyl‐2‐acetamido‐2‐deoxy‐α‐ d ‐galactopyranoside (BAGN), an inhibitor of O‐glycosylation commonly used in various cell lines, resulted in an elevated proportion of HIV‐infected cells, an increased quantity of HIV p24 protein per cell, and higher levels of viral particles in supernatants. This finding could potentially offer new perspectives on therapeutic targets related to O‐glycosylation for strategies aimed at controlling HIV 112 .…”