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The Role of Crude Human Saliva and Purified Salivary MUC5B and MUC7 Mucins in the Inhibition of Human Immunodeficiency Virus Type 1 in an Inhibition Assay

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The Role of Crude Human Saliva and Purified Salivary MUC5B and MUC7 Mucins in the Inhibition of Human Immunodeficiency Virus Type 1 in an Inhibition Assay

Habtom H Habte et al. Virol J.

Abstract

Background: Despite the continuous shedding of HIV infected blood into the oral cavity and the detectable presence of the AIDS virus at a high frequency, human saliva is reported to inhibit oral transmission of HIV through kissing, dental treatment, biting, and aerosolization. The purpose of this study was to purify salivary MUC5B and MUC7 mucins from crude saliva and determine their anti-HIV-1 activities.

Methods: Following Sepharose CL-4B column chromatography and caesium chloride isopycnic density-gradient ultra-centrifugation, the purity and identity of the mucins was determined by SDS-PAGE and Western blotting analysis respectively. Subsequently an HIV-1 inhibition assay was carried out to determine the anti-HIV-1 activity of the crude saliva and purified salivary mucins by incubating them with subtype D HIV-1 prior to infection of the CD4+ CEM SS cells.

Results: Western blotting analysis confirmed that the mucin in the void volume is MUC5B and the mucin in the included volume is MUC7. The HIV inhibition assay revealed that both the crude saliva and salivary MUC5B and MUC7 mucins inhibited HIV-1 activity by 100%.

Conclusion: Although the mechanism of action is not clear the carbohydrate moieties of the salivary mucins may trap or aggregate the virus and prevent host cell entry.

Figures

Figure 1
Figure 1
Sepharose CL-4B gel filtration of crude saliva. An aliquot (20 ml) of soluble human crude saliva extracted in 6 M GuHCl containing 10 mM EDTA, 5 mM NEM, 1 mM PMSF and 0.1% CHAPS pH 6.5 was chromatographed and eluted with 4 M GuHCl containing 10 mM EDTA, 5 mM NEM and 0.05% CHAPS pH 6.5 at flow rate of 48 ml/h at room temperature. Fractions were analysed for carbohydrate with PAS at 555 nm (◆) and for protein A280 (■). Materials eluted in the void volume (Vo) and included volume (Vi) were pooled separately, dialysed against three changes of distilled water for overnight at 4°C and freeze-dried.
Figure 2
Figure 2
SDS-PAGE analysis of the salivary mucus eluted in the Vo of the Sepharose CL-4B gel filtration column. Freeze-dried material (30 μg) of the Vo prepared in reducing gel loading buffer was separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue G-250 (a) and PAS (b). The molecular weight marker is indicated by Mw and lanes 1 to 6 represents samples from 6 donors.
Figure 3
Figure 3
SDS-PAGE analysis of the salivary mucus eluted in the Vi of the Sepharose CL-4B gel filtration column. Freeze-dried material (30 μg) of the Vi prepared in reducing gel loading buffer was separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue G-250 (a) and PAS (b). The molecular weight marker is indicated by Mw and lanes 1 to 6 represents samples from 6 donors.
Figure 4
Figure 4
Caesium chloride isopycnic density-gradient ultra-centrifugation of the material eluted in the Vi of the gel filtration column. Freeze-dried material eluted in the Vi was dissolved in 4 M GuHCl containing 10 mM EDTA, 5 mM NEM and 0.05% CHAPS pH 6.5 and adjusted to a density of 1.39 to 1.40 g/ml with caesium chloride. Density gradient centrifugation was performed in a Beckman L45 ultra-centrifuge for 48 h at 105 000 g at 4°C. Fractions with high PAS (◆) and low protein (Lowry) (■) (a) were pooled and adjust to a density of 1.39 to 1.40 g/ml with caesium chloride for a second step centrifugation (b). Mucin rich fractions with a density (▲) between 1.37–1.42 were pooled, dialysed against three changes of distilled water for overnight at 4°C and freeze-dried.
Figure 5
Figure 5
SDS-PAGE analysis of the purified Vi material. After purification, mucins were loaded onto a 10% SDS-PAGE and stained with Coomassie Brilliant Blue G-250 (lane, 1 and 2) and PAS (lane, 3). Molecular weight markers and the two mucin isoforms are indicated by lane 1and the arrows respectively.
Figure 6
Figure 6
Western blotting analysis of Vo and Vi material from the gel filtration column using rabbit anti-MUC5B and goat anti-MUC7 polyclonal antibodies. a) Lanes 1, MUC7 (negative control), 2 and 3, Vo material from two donors and 4, respiratory MUC5B (positive control) and in b) Lanes 1, crude saliva (positive control), 2, gastric mucin (negative control) and 3, purified Vi material were separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. Following overnight blocking, the membranes were incubated for 2 h with rabbit anti-MUC5B (a) and goat anti-MUC7 (b) polyclonal antibodies. Membranes were then incubated for 1 h with goat anti-rabbit (a) and rabbit anti-goat (b) HRPO linked secondary antibodies and bands that interacted with the antibodies were detected by exposing the membrane to ECL detection. The arrows indicate the two glycoforms of MUC7.
Figure 7
Figure 7
Inhibition of HIV-1 activity by human crude saliva and purified salivary MUC5B and MUC7 mucins. Crude saliva and purified salivary MUC5B and MUC7 mucins (0.9 mg each) were incubated with subtype D HIV-1 for 60 min and filtered through 0.45 μm pore size cellulose acetate filter. As controls HIV-1 treated with media and heat inactivated HIV-1 were used. The unfiltered samples were then incubated with CD4+ CEM SS cells at a concentration of 0.5 × 106cells/ml for 30 min, 1 h and 3 h. After PBS wash cells were cultured and viral replication was measured by a qualitative p24 antigen assay.
Figure 8
Figure 8
Inhibition of HIV-1 activity by human crude saliva and purified salivary MUC5B and MUC7 mucins. Crude saliva and purified salivary MUC5B and MUC7 mucins (0.9 mg each) were incubated with subtype D HIV-1 for 60 min and filtered through 0.45 μpore size cellulose acetate filter. As controls HIV-1 treated with media and heat inactivated HIV-1 were used. The filtrates of the mixtures were then incubated with CD4+ CEM SS cells at a concentration of 0.5 × 106cells/ml for 30min, 1h and 3h. After PBS wash cells were cultured and viral replication was measured by a qualitative p24 antigen assay.

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