Differential sensitivity of HIV-1 viruses to lectins.

<p>Differential sensitivity of HIV-1 viruses to lectins.</p

Glycan biosynthetic pathway and its inhibitors.

<p>N-linked glycans are transferred <i>en bloc</i> onto the nascent peptide after the peptide emerges from the ribosome in the endoplasmic reticulum (ER). The immature high-mannose structure is trimmed by glycosidases and subsequently processed to form hybrid- and complex-type glycans. Kifunensine is a drug inhibitor of the ER and Golgi mannosidase I, thus arresting glycosylation at Man₉GlcNAc₂. Production of glycoproteins in GnTI-deficient cells, on the other hand, resulted in accumulation of the Man₅GlcNAc₂ structure. Swainsonine inhibits mannosidase II in the Golgi that is required for the maturation of high mannose and hybrid glycans into complex glycans.</p

Effect of <i>N</i>-glycan modification on virus sensitivity to lectins.

<p>JRFL.JB and REJO4541.67 viruses were produced in 293T cells in the presence of glycosylation-pathway inhibitors kifunensine or swainsonine, or in 293S GnTI^-/- cells lacking GlcNAc transferase I. Lectins were serially diluted; viruses were then added at 200 TCID₅₀ and incubated for 1 h at 37°C before addition of TZM-bl cells. After 48 h, virus infection was measured based on β-galactosidase activity. Cells infected with viruses only, in the absence of lectins, represent 100% infection. Percentage of virus inhibition by lectin was calculated using virus control (virus and cells only, 0% inhibition) and cell control (cell and no virus, 100% inhibition). All experiments were performed in duplicate and repeated three times. Data are shown as the averages and standard deviations from all 3 experiments.</p

Effect of <i>N</i>-glycan modification on Env incorporation to virions.

<p>Viruses were produced in 293T HEK cells in the presence or absence of glycosidase inhibitors kifunensine and swainsonine or in 293S HEK GnTI-/- cells. A) JRFL virus preparations were loaded to SDS-PAGE after normalization for CA-p24 content. Env was detected by Western blot with anti-gp120 mAbs and quantified using Image Lab (BioRad) software. B) The relative contents of Env associated with N-glycan-modified JRFL viruses were calculated in comparison with Env of wild type control (set at 100%). C) The relative Env incorporation to N-glycan modified vs wild type REJO viruses. Data represent averages and standard errors from 2 experiments (each in duplicate) and were analyzed using unpaired t-test. ***p <0.0005, ** p <0.005. D) Correlation of Env incorporation to virions and virus infectivity by spearman’s rank test.</p

Deglycosylation of wild type vs glycan-modified Envs by Endo-H and PNGase F.

<p>a) JRFL viruses were produced in 293T cells with kifunensine or swansonine or in GnTI-/- cells, concentrated from culture supernatants by Lenti X-100, and then treated (+) or not treated (-) with Endo H to remove high-mannose and hybrid-mannose <i>N</i>-glycans but not complex <i>N</i>-glycans. b) JRFL viruses were similarly produced and treated with (+) or without (-) PNGase F to remove all <i>N</i>-glycans. The digestion products were subjected to SDS-PAGE under a reduced condition and Western blot analysis with anti-gp120 mAbs. Untreated Env gp120 and enzyme treated Env gp120 are indicated by black and red arrows, respectively.</p

Effect of <i>N</i>-glycan modification on HIV-1 infectivity.

<p>A) Viruses produced in 293T cells with different glycosidase inhibitors (kifunensine or swansonine) or in 293S GnTI^-/- cells were titrated as a function of CA-p24 content and their infectivity in TZM-bl reporter cells was measured after 48 h by β-galactosidase activity. B) Statistical analyses were performed to compare infectivity of the different viruses when the same virus input (5 ng/mL CA-p24) was used to infect TZM-bl cells. p24 concentration was measured by ELISA. Averages and standard errors from 2 independent experiments (each performed in duplicate) are shown and analyzed using unpaired t-test. **p < 0.005; ***p < 0.0005.</p

Mannan competition assay.

<p>GRFT and GNA were serially 2-fold diluted starting from 10μg and 250μg, respectively, to attain approximately 100% virus inhibition at all concentrations tested. When mannan (10 μg /mL) was used, it was added to lectins, and the mixtures were incubated with wild type or glycan-modified JRFL viruses (200 TCID₅₀) for 1 h at 37°C, before addition of TZM-bl cells. After 48 h, virus infection was measured based on β-galactosidase activity. All experiments were performed in duplicate and repeated twice. Data are shown as averages and standard deviations from the 2 experiments.</p

Effects of <i>N</i>-glycan modification on Env molecular mass and virus infectivity.

<p>Viruses were produced by transient transfection of 293S GnTI^-/- cells or 293T cells in the presence or absence of kifunensine or swainsonine. A) Relative molecular weight of Env as measured by SDS-PAGE (reduced condition) and Western blot. Oligosaccharide compositions predominant in the different glycan-modified virus preparations are indicated below the blots. Cleaved Env gp120 and uncleaved Env gp160 are indicated by red and black arrows, respectively. B) Production of REJO4541.67 and JRFL.JB viruses under the different conditions. Culture supernatants containing viruses were serially diluted 2-fold and added to TZM-bl cells. After 48 h, cells were lysed, and β-galactosidase activity was measured with a luminescence substrate. Data represent averages and standard deviations of 2 independent experiments.</p

Lectins and their oligosaccharide and N-glycan type specificity.

<p>Lectins and their oligosaccharide and N-glycan type specificity.</p

Virus inhibition by lectins that differ in oligosaccharide specificity.

<p>Lectins were tested to block infection of HIV-1 pseudoviruses SF162.LS (tier 1A), Bal.01 (tier 1B), JRFL.JB (tier 2, chronic), and REJO4541.67 (tier 2, acute) in TZM-bl cells. Lectins were serially diluted, added to viruses (200 TCID₅₀), and incubated for 1 h at 37°C before addition of TZM-bl cells. After 48 h, virus infection was measured by β-galactosidase activity. Cell viability was measured in parallel using CellTiter Glow kit (Promega). Experiments were performed four times; averages and standard deviations from all experiments are shown.</p