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Rhadinovirus host entry by co-operative infection.

Lawler C, Milho R, May JS, Stevenson PG - PLoS Pathog. (2015)

Bottom Line: Although epithelial cell lines endocytose MuHV-4 readily in vitro, AECs did not.An antibody block of epithelial cell binding failed to block host entry: opsonization provided merely another route to macrophages.By contrast an antibody block of membrane fusion was effective.

View Article: PubMed Central - PubMed

Affiliation: Sir Albert Sakzewski Virus Research Centre, School of Chemistry and Molecular Biosciences, Royal Children's Hospital and University of Queensland, Brisbane, Australia.

ABSTRACT
Rhadinoviruses establish chronic infections of clinical and economic importance. Several show respiratory transmission and cause lung pathologies. We used Murid Herpesvirus-4 (MuHV-4) to understand how rhadinovirus lung infection might work. A primary epithelial or B cell infection often is assumed. MuHV-4 targeted instead alveolar macrophages, and their depletion reduced markedly host entry. While host entry was efficient, alveolar macrophages lacked heparan - an important rhadinovirus binding target - and were infected poorly ex vivo. In situ analysis revealed that virions bound initially not to macrophages but to heparan⁺ type 1 alveolar epithelial cells (AECs). Although epithelial cell lines endocytose MuHV-4 readily in vitro, AECs did not. Rather bound virions were acquired by macrophages; epithelial infection occurred only later. Thus, host entry was co-operative - virion binding to epithelial cells licensed macrophage infection, and this in turn licensed AEC infection. An antibody block of epithelial cell binding failed to block host entry: opsonization provided merely another route to macrophages. By contrast an antibody block of membrane fusion was effective. Therefore co-operative infection extended viral tropism beyond the normal paradigm of a target cell infected readily in vitro; and macrophage involvement in host entry required neutralization to act down-stream of cell binding.

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Heparan expression in lungs.A Lungs of naive mice were stained for PDP (green in merge), CD68 (white in merge), and sulfated heparan with mAb 10E4 (red in merge). Nuclei were stained with DAPI (blue). Two example images are shown, representative of 10 sections from 2 mice. Heparan co-localized with PDP (type 1 AECs) and not with CD68 (alveolar macrophages). B Lung sections as in a were stained for PDP, CD68, and unsulfated heparan with mAb NAH46 (red in merge). Two example images are shown, representative of 10 sections from 2 mice. Heparan co-localized with PDP and not with CD68. C Lung sections as in a were stained for PDP, collagen IV (white in merge), and sulfated heparan with mAb 10E4. Heparan and collagen IV showed equivalent distributions. Images are representative of 8 sections from 2 mice. D Lung sections as in a were stained for PDP, collagen IV, and unsulfated heparan with mAb NAH46. Again heparan and collagen IV showed extensive co-localization, but some NAH46+PDP+ regions were collagen IV- (arrows). These abutted alveolar air spaces, and so correspond to the apical surface of type 1 AECs.
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ppat.1004761.g006: Heparan expression in lungs.A Lungs of naive mice were stained for PDP (green in merge), CD68 (white in merge), and sulfated heparan with mAb 10E4 (red in merge). Nuclei were stained with DAPI (blue). Two example images are shown, representative of 10 sections from 2 mice. Heparan co-localized with PDP (type 1 AECs) and not with CD68 (alveolar macrophages). B Lung sections as in a were stained for PDP, CD68, and unsulfated heparan with mAb NAH46 (red in merge). Two example images are shown, representative of 10 sections from 2 mice. Heparan co-localized with PDP and not with CD68. C Lung sections as in a were stained for PDP, collagen IV (white in merge), and sulfated heparan with mAb 10E4. Heparan and collagen IV showed equivalent distributions. Images are representative of 8 sections from 2 mice. D Lung sections as in a were stained for PDP, collagen IV, and unsulfated heparan with mAb NAH46. Again heparan and collagen IV showed extensive co-localization, but some NAH46+PDP+ regions were collagen IV- (arrows). These abutted alveolar air spaces, and so correspond to the apical surface of type 1 AECs.

Mentions: To identify heparan+ cells in the lungs we stained sections with mAb 10E4, which recognizes sulfated heparan [50] (Fig. 6A), and with mAb NAH46, which recognizes non-sulfated heparan [51] (Fig. 6B). Staining by both mAbs co-localized with PDP (type 1 AECs) and not with CD68 (alveolar macrophages). To distinguish apical from basolateral heparan, we co-stained sections for the alveolar basement membrane component collagen IV (Fig. 6C). Sulfated heparan (mAb 10E4) co-localized completely with collagen IV and so was just basolateral. Unsulfated heparan (mAb NAH46) also co-localized largely with collagen IV. However there were also NAH46+ surfaces abutting the air spaces that lacked collagen IV. Therefore specifically unsulfated heparan was accessible to incoming virions on the apical AEC surface.


Rhadinovirus host entry by co-operative infection.

Lawler C, Milho R, May JS, Stevenson PG - PLoS Pathog. (2015)

Heparan expression in lungs.A Lungs of naive mice were stained for PDP (green in merge), CD68 (white in merge), and sulfated heparan with mAb 10E4 (red in merge). Nuclei were stained with DAPI (blue). Two example images are shown, representative of 10 sections from 2 mice. Heparan co-localized with PDP (type 1 AECs) and not with CD68 (alveolar macrophages). B Lung sections as in a were stained for PDP, CD68, and unsulfated heparan with mAb NAH46 (red in merge). Two example images are shown, representative of 10 sections from 2 mice. Heparan co-localized with PDP and not with CD68. C Lung sections as in a were stained for PDP, collagen IV (white in merge), and sulfated heparan with mAb 10E4. Heparan and collagen IV showed equivalent distributions. Images are representative of 8 sections from 2 mice. D Lung sections as in a were stained for PDP, collagen IV, and unsulfated heparan with mAb NAH46. Again heparan and collagen IV showed extensive co-localization, but some NAH46+PDP+ regions were collagen IV- (arrows). These abutted alveolar air spaces, and so correspond to the apical surface of type 1 AECs.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4366105&req=5

ppat.1004761.g006: Heparan expression in lungs.A Lungs of naive mice were stained for PDP (green in merge), CD68 (white in merge), and sulfated heparan with mAb 10E4 (red in merge). Nuclei were stained with DAPI (blue). Two example images are shown, representative of 10 sections from 2 mice. Heparan co-localized with PDP (type 1 AECs) and not with CD68 (alveolar macrophages). B Lung sections as in a were stained for PDP, CD68, and unsulfated heparan with mAb NAH46 (red in merge). Two example images are shown, representative of 10 sections from 2 mice. Heparan co-localized with PDP and not with CD68. C Lung sections as in a were stained for PDP, collagen IV (white in merge), and sulfated heparan with mAb 10E4. Heparan and collagen IV showed equivalent distributions. Images are representative of 8 sections from 2 mice. D Lung sections as in a were stained for PDP, collagen IV, and unsulfated heparan with mAb NAH46. Again heparan and collagen IV showed extensive co-localization, but some NAH46+PDP+ regions were collagen IV- (arrows). These abutted alveolar air spaces, and so correspond to the apical surface of type 1 AECs.
Mentions: To identify heparan+ cells in the lungs we stained sections with mAb 10E4, which recognizes sulfated heparan [50] (Fig. 6A), and with mAb NAH46, which recognizes non-sulfated heparan [51] (Fig. 6B). Staining by both mAbs co-localized with PDP (type 1 AECs) and not with CD68 (alveolar macrophages). To distinguish apical from basolateral heparan, we co-stained sections for the alveolar basement membrane component collagen IV (Fig. 6C). Sulfated heparan (mAb 10E4) co-localized completely with collagen IV and so was just basolateral. Unsulfated heparan (mAb NAH46) also co-localized largely with collagen IV. However there were also NAH46+ surfaces abutting the air spaces that lacked collagen IV. Therefore specifically unsulfated heparan was accessible to incoming virions on the apical AEC surface.

Bottom Line: Although epithelial cell lines endocytose MuHV-4 readily in vitro, AECs did not.An antibody block of epithelial cell binding failed to block host entry: opsonization provided merely another route to macrophages.By contrast an antibody block of membrane fusion was effective.

View Article: PubMed Central - PubMed

Affiliation: Sir Albert Sakzewski Virus Research Centre, School of Chemistry and Molecular Biosciences, Royal Children's Hospital and University of Queensland, Brisbane, Australia.

ABSTRACT
Rhadinoviruses establish chronic infections of clinical and economic importance. Several show respiratory transmission and cause lung pathologies. We used Murid Herpesvirus-4 (MuHV-4) to understand how rhadinovirus lung infection might work. A primary epithelial or B cell infection often is assumed. MuHV-4 targeted instead alveolar macrophages, and their depletion reduced markedly host entry. While host entry was efficient, alveolar macrophages lacked heparan - an important rhadinovirus binding target - and were infected poorly ex vivo. In situ analysis revealed that virions bound initially not to macrophages but to heparan⁺ type 1 alveolar epithelial cells (AECs). Although epithelial cell lines endocytose MuHV-4 readily in vitro, AECs did not. Rather bound virions were acquired by macrophages; epithelial infection occurred only later. Thus, host entry was co-operative - virion binding to epithelial cells licensed macrophage infection, and this in turn licensed AEC infection. An antibody block of epithelial cell binding failed to block host entry: opsonization provided merely another route to macrophages. By contrast an antibody block of membrane fusion was effective. Therefore co-operative infection extended viral tropism beyond the normal paradigm of a target cell infected readily in vitro; and macrophage involvement in host entry required neutralization to act down-stream of cell binding.

Show MeSH
Related in: MedlinePlus