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The mannose receptor mediates dengue virus infection of macrophages.

Miller JL, de Wet BJ, deWet BJ, Martinez-Pomares L, Radcliffe CM, Dwek RA, Rudd PM, Gordon S - PLoS Pathog. (2008)

Bottom Line: To our knowledge, we show for the first time that the MØ mannose receptor (MR) binds to all four serotypes of DV and specifically to the envelope glycoprotein.This binding is abrogated by deglycosylation of the DV envelope glycoprotein.Surface expression of recombinant MR on NIH3T3 cells confers DV binding.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Macrophages (MØ) and mononuclear phagocytes are major targets of infection by dengue virus (DV), a mosquito-borne flavivirus that can cause haemorrhagic fever in humans. To our knowledge, we show for the first time that the MØ mannose receptor (MR) binds to all four serotypes of DV and specifically to the envelope glycoprotein. Glycan analysis, ELISA, and blot overlay assays demonstrate that MR binds via its carbohydrate recognition domains to mosquito and human cell-produced DV antigen. This binding is abrogated by deglycosylation of the DV envelope glycoprotein. Surface expression of recombinant MR on NIH3T3 cells confers DV binding. Furthermore, DV infection of primary human MØ can be blocked by anti-MR antibodies. MR is a prototypic marker of alternatively activated MØ, and pre-treatment of human monocytes or MØ with type 2 cytokines (IL-4 or IL-13) enhances their susceptibility to productive DV infection. Our findings indicate a new functional role for the MR in DV infection.

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Anti-MR and Anti-DC-SIGN Antibodies Inhibit the Ability of DV2 to Infect IL-4-Treated Monocytes(A) IL-4-treated human monocytes were incubated in triplicate wells with medium alone (no block), 2mg/ml mannan, titrations of goat anti-human MR antiserum, or with normal goat serum (NGS). Treated cells were infected with mosquito cell–grown 16681 DV2 at a multiplicity of infection of 0.5 in the presence of these inhibitors, incubated for 48 h and fixed.(B) Monocytes were treated in triplicate wells, as above, including blocking with monoclonal antibodies specific for DC-SIGN (120507), DC-SIGNR (120604), and both DC-SIGN and DC-SIGNR (DC28 and 120612) or isotype controls (all at 5ug/ml) prior to infection with mosquito cell–grown 16681 DV2 at a multiplicity of infection of 0.04 in the presence of these inhibitors. Following fixation, cells were immunolabelled with anti-DV E protein monoclonal antibody 3H5 and counted using a fluorescent microscope, and percent of cells infected calculated. Data are expressed as mean and SD of triplicate wells. Representative data from one of multiple donors are shown.(C) The titre of infectious virus in the cell supernatant at 48 h post infection was determined by plaque assay. Due to variation between donors, the titre at 48 h in the absence of block was normalised to 100. Each data point is the average of three infected wells, each plaqued in triplicate, and the results from six donors are shown in this graph (mean represented by a bar). The p value was calculated by unpaired, two-tailed t test.
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ppat-0040017-g007: Anti-MR and Anti-DC-SIGN Antibodies Inhibit the Ability of DV2 to Infect IL-4-Treated Monocytes(A) IL-4-treated human monocytes were incubated in triplicate wells with medium alone (no block), 2mg/ml mannan, titrations of goat anti-human MR antiserum, or with normal goat serum (NGS). Treated cells were infected with mosquito cell–grown 16681 DV2 at a multiplicity of infection of 0.5 in the presence of these inhibitors, incubated for 48 h and fixed.(B) Monocytes were treated in triplicate wells, as above, including blocking with monoclonal antibodies specific for DC-SIGN (120507), DC-SIGNR (120604), and both DC-SIGN and DC-SIGNR (DC28 and 120612) or isotype controls (all at 5ug/ml) prior to infection with mosquito cell–grown 16681 DV2 at a multiplicity of infection of 0.04 in the presence of these inhibitors. Following fixation, cells were immunolabelled with anti-DV E protein monoclonal antibody 3H5 and counted using a fluorescent microscope, and percent of cells infected calculated. Data are expressed as mean and SD of triplicate wells. Representative data from one of multiple donors are shown.(C) The titre of infectious virus in the cell supernatant at 48 h post infection was determined by plaque assay. Due to variation between donors, the titre at 48 h in the absence of block was normalised to 100. Each data point is the average of three infected wells, each plaqued in triplicate, and the results from six donors are shown in this graph (mean represented by a bar). The p value was calculated by unpaired, two-tailed t test.

Mentions: The functional role of MR in DV infection of primary human MØ was investigated using a polyclonal anti-MR antibody to block infection. This was examined in the IL-4-treated MDMØ since these cells showed the highest rate of DV infection. Anti-MR antibody significantly blocked DV infection of IL-4-treated MDMØ (p = 0.008) in all donors tested (6 donors; Figure 7A shows data from one representative donor). Normal goat serum control did not inhibit infection, suggesting that the MR may be a new functional receptor contributing to DV infection of human MØ. Production of infectious virus (pfu/ml) by these cells at 2 d post infection was reduced by 60%–95% with either mannan or anti-human MR antibody (Figure 7C), indicating that attachment and/or entry via this receptor is required for productive infection. The goat anti-human MR antibody blocked mannosylated BSA-FITC binding to both IL-4-treated MDMØ and 3T3 cells transfected with human MR (Figure S1).


The mannose receptor mediates dengue virus infection of macrophages.

Miller JL, de Wet BJ, deWet BJ, Martinez-Pomares L, Radcliffe CM, Dwek RA, Rudd PM, Gordon S - PLoS Pathog. (2008)

Anti-MR and Anti-DC-SIGN Antibodies Inhibit the Ability of DV2 to Infect IL-4-Treated Monocytes(A) IL-4-treated human monocytes were incubated in triplicate wells with medium alone (no block), 2mg/ml mannan, titrations of goat anti-human MR antiserum, or with normal goat serum (NGS). Treated cells were infected with mosquito cell–grown 16681 DV2 at a multiplicity of infection of 0.5 in the presence of these inhibitors, incubated for 48 h and fixed.(B) Monocytes were treated in triplicate wells, as above, including blocking with monoclonal antibodies specific for DC-SIGN (120507), DC-SIGNR (120604), and both DC-SIGN and DC-SIGNR (DC28 and 120612) or isotype controls (all at 5ug/ml) prior to infection with mosquito cell–grown 16681 DV2 at a multiplicity of infection of 0.04 in the presence of these inhibitors. Following fixation, cells were immunolabelled with anti-DV E protein monoclonal antibody 3H5 and counted using a fluorescent microscope, and percent of cells infected calculated. Data are expressed as mean and SD of triplicate wells. Representative data from one of multiple donors are shown.(C) The titre of infectious virus in the cell supernatant at 48 h post infection was determined by plaque assay. Due to variation between donors, the titre at 48 h in the absence of block was normalised to 100. Each data point is the average of three infected wells, each plaqued in triplicate, and the results from six donors are shown in this graph (mean represented by a bar). The p value was calculated by unpaired, two-tailed t test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233670&req=5

ppat-0040017-g007: Anti-MR and Anti-DC-SIGN Antibodies Inhibit the Ability of DV2 to Infect IL-4-Treated Monocytes(A) IL-4-treated human monocytes were incubated in triplicate wells with medium alone (no block), 2mg/ml mannan, titrations of goat anti-human MR antiserum, or with normal goat serum (NGS). Treated cells were infected with mosquito cell–grown 16681 DV2 at a multiplicity of infection of 0.5 in the presence of these inhibitors, incubated for 48 h and fixed.(B) Monocytes were treated in triplicate wells, as above, including blocking with monoclonal antibodies specific for DC-SIGN (120507), DC-SIGNR (120604), and both DC-SIGN and DC-SIGNR (DC28 and 120612) or isotype controls (all at 5ug/ml) prior to infection with mosquito cell–grown 16681 DV2 at a multiplicity of infection of 0.04 in the presence of these inhibitors. Following fixation, cells were immunolabelled with anti-DV E protein monoclonal antibody 3H5 and counted using a fluorescent microscope, and percent of cells infected calculated. Data are expressed as mean and SD of triplicate wells. Representative data from one of multiple donors are shown.(C) The titre of infectious virus in the cell supernatant at 48 h post infection was determined by plaque assay. Due to variation between donors, the titre at 48 h in the absence of block was normalised to 100. Each data point is the average of three infected wells, each plaqued in triplicate, and the results from six donors are shown in this graph (mean represented by a bar). The p value was calculated by unpaired, two-tailed t test.
Mentions: The functional role of MR in DV infection of primary human MØ was investigated using a polyclonal anti-MR antibody to block infection. This was examined in the IL-4-treated MDMØ since these cells showed the highest rate of DV infection. Anti-MR antibody significantly blocked DV infection of IL-4-treated MDMØ (p = 0.008) in all donors tested (6 donors; Figure 7A shows data from one representative donor). Normal goat serum control did not inhibit infection, suggesting that the MR may be a new functional receptor contributing to DV infection of human MØ. Production of infectious virus (pfu/ml) by these cells at 2 d post infection was reduced by 60%–95% with either mannan or anti-human MR antibody (Figure 7C), indicating that attachment and/or entry via this receptor is required for productive infection. The goat anti-human MR antibody blocked mannosylated BSA-FITC binding to both IL-4-treated MDMØ and 3T3 cells transfected with human MR (Figure S1).

Bottom Line: To our knowledge, we show for the first time that the MØ mannose receptor (MR) binds to all four serotypes of DV and specifically to the envelope glycoprotein.This binding is abrogated by deglycosylation of the DV envelope glycoprotein.Surface expression of recombinant MR on NIH3T3 cells confers DV binding.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Macrophages (MØ) and mononuclear phagocytes are major targets of infection by dengue virus (DV), a mosquito-borne flavivirus that can cause haemorrhagic fever in humans. To our knowledge, we show for the first time that the MØ mannose receptor (MR) binds to all four serotypes of DV and specifically to the envelope glycoprotein. Glycan analysis, ELISA, and blot overlay assays demonstrate that MR binds via its carbohydrate recognition domains to mosquito and human cell-produced DV antigen. This binding is abrogated by deglycosylation of the DV envelope glycoprotein. Surface expression of recombinant MR on NIH3T3 cells confers DV binding. Furthermore, DV infection of primary human MØ can be blocked by anti-MR antibodies. MR is a prototypic marker of alternatively activated MØ, and pre-treatment of human monocytes or MØ with type 2 cytokines (IL-4 or IL-13) enhances their susceptibility to productive DV infection. Our findings indicate a new functional role for the MR in DV infection.

Show MeSH
Related in: MedlinePlus