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.

Keywords: O-glycosylation; benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside; human immunodeficiency virus-1; infectivity; replication; viral outgrowth.

Figure 1

Benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (BAGN) effect on NL4-3 production is dose dependent and pretreatment of target cells increases infection rates. (A) Cells were infected with NL4-3 at a multiplicity of infection of 0.01 and cultured for 5 days. Cell viability, number of p24 positive CD4 T cells, and the median fluorescence intensity (MFI) of p24-specific staining are shown for one representative experiment. (B) Same as in panel (A) except that target cells were pre-incubated with 2 mM of BAGN overnight before infecting them.

Figure 2

Synergistic effect on human immunodeficiency virus (HIV) production of Benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (BAGN)-grown virus and culturing target cells with BAGN. (A) Experimental outline for generating BAGN-treated/untreated cells (left half), growing virus in the presence/absence of BAGN (right half), and performing the viral replication assay (VRA, central square). (B) The number of p24+ CD4 T cells is shown depending on the different culture conditions, median and interquartile ranges are indicated by bars (N = 10). BAGN-grown virus represents virus stocks previously grown in the presence of BAGN. BAGN-treated cells refer to pretreatment and continuous presence of BAGN in the culture. Panels (C,D) are same as in panel (B) showing the p24 in the supernatant and the median fluorescence intensity (MFI) of infected cells, respectively. Mann–Whitney significance level are indicated by *p < 0.05, **p < 0.001, ***p < 0.0001.

Figure 3

Benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (BAGN) exposure modulates CD25, HLA-DR, and CCR5 expression. The expression of (A) CD4 activation marker and (B) human immunodeficiency virus receptors CXCR4 and CCR5 on PHA-activated CD4 T cells was assessed by flow cytometry and expressed as either the percentage of positive cells or median fluorescence intensity (MFI). Open circles or squares indicate cells grown in the absence of BAGN, filled circles or squares show cells grown in the presence of BAGN. Median and interquartile ranges are indicated by bars (N = 20). (C) Correlation between levels of expression of activation markers and CCR5 co-receptor on BAGN-treated PHA-blasts (MFI) and the number of infected cells (measured as % of CD3+ CD8− p24+ cells). Mann–Whitney significance level are indicated by *p < 0.05, **p < 0.001, ***p < 0.0001.

Figure 4

Benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (BAGN) increases recovery rate in viral outgrowth assays. Peripheral blood mononuclear cells from 12 human immunodeficiency virus-infected individuals were cocultured with PHA-blasts, pretreated and cultured, or not, in BAGN containing medium. Virus recovery failed in four subjects, independent of the culture condition (not shown). In four subjects (A), virus recovery was only possible in the presence of BAGN, and in another four individuals (B), culture with BAGN yielded virus more rapidly and to higher levels compared to cultures without BAGN. (C) Virus grown either with or without BAGN conditions was sequenced (envelope V3) and clustered by neighbor-joining tree analysis for the four individuals shown in panel (B). Squares indicate the different sequences, in green those obtained without BAGN in red with BAGN. Intensity of the color indicates relative frequency of the sequence. Sequences predicted to be of X4 tropism are indicated by *.

Figure 5

Benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (BAGN) improves outgrowth of latent virus but only in the presence of latency-reversing agents. ACH2 cells were incubated with DMSO or with panobinostat/bryostatin with increasing doses of BAGN. Virus outgrowth was measured at 24 h by determining the TCID50/ml in duplicates in TZM-bl cells.