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. 2010 Jan 12;107(2):866-71.
doi: 10.1073/pnas.0913351107. Epub 2009 Dec 22.

Myelin-associated Glycoprotein Mediates Membrane Fusion and Entry of Neurotropic Herpesviruses

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Free PMC article

Myelin-associated Glycoprotein Mediates Membrane Fusion and Entry of Neurotropic Herpesviruses

Tadahiro Suenaga et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Varicella-zoster virus (VZV) and herpes simplex virus (HSV) are prevalent neurotropic herpesviruses that cause various nervous system diseases. Similar to other enveloped viruses, membrane fusion is an essential process for viral entry. Therefore, identification of host molecules that mediate membrane fusion is important to understand the mechanism of viral infection. Here, we demonstrate that myelin-associated glycoprotein (MAG), mainly distributed in neural tissues, associates with VZV glycoprotein B (gB) and promotes cell-cell fusion when coexpressed with VZV gB and gH/gL. VZV preferentially infected MAG-transfected oligodendroglial cells. MAG also associated with HSV-1 gB and enhanced HSV-1 infection of promyelocytes. These findings suggested that MAG is involved in VZV and HSV infection of neural tissues.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Association of MAG with VZV-gB. (A) HSV-gB- (Upper, bold lines) and VZV-gB- (Lower, bold lines) transfected 293T cells were stained with control-Ig and PILRα-Ig, as well as anti-HSV-gB or anti-VZV-gB mAb. Mock-transfectants were used as control (thin lines). (B) 293T cells were transfected with MAG and PILRα (bold line), and these cells, as well as control-transfectants (thin line), were stained with VZV-gB-Ig. (C) 293T cells transfected with VZV-glycoproteins (bold line) and control-transfectants (thin line) were stained with MAG-Ig.
Fig. 2.
Fig. 2.
Association of MAG with gB and gE expressed in VZV-infected cells. (A) VZV-infected MeWo (bold line) or mock-infected MeWo cells (thin line) were stained with anti-VZV-gB mAb, anti-VZV-gE mAb, MAG-Ig, or control-Ig, respectively. (B) Association of MAG with gB and gE expressed in VZV-infected cells. Cell lysates of VZV-infected MeWo or mock-infected MeWo cells were immunoprecipitated with control-Ig or MAG-Ig and the precipitants were analyzed by Western blotting using anti-VZV-gB, anti-VZV-gE, or anti-VZV-gH mAbs. Uncleaved and cleaved forms of gB were indicated in the figure. Data are representative of three independent experiments.
Fig. 3.
Fig. 3.
Cell-cell fusion induced by MAG (A) MAG but not IDE mediates cell-cell fusion by associating with VZV-glycoproteins. 293T cells transfected with VZV-gB, gE, gH, gL, and GFP and 293T cells transfected with MAG and IDE, as well as DsRed (Target), were cocultured for 18 h and were analyzed by fluorescence microscopy. Green and red fluorescence from nonfused cells were converted to gray color, and yellow colors of fused cells were left unchanged (processed color). Cell nuclei were stained with Hoechst 33258 fluorescence dye, and blue fluorescence from nuclei is shown (gray). Fused cells are delineated by red lines. Mock-transfectants were used as control (Cont.). When mock- or IDE-transfectants were cocultured, number of obvious polykaryons in each field of microscopy was 0.5 ± 0.6 or 0.5 ± 0.7 (mean ± SD of 20 fields), respectively. On the other hand, when MAG transfectants were cocultured, the number of obvious polykaryons was 5.7 ± 1.5. (B) Quantification of efficiency of MAG-mediated membrane fusion. 293T cells transfected with VZV-gB, gE, gH, gL, and T7 polymerase and 293T cells transfected with MAG and IDE, as well as firefly luciferase and Renilla luciferase, were cocultured for 18 h. (C) MPRci does not mediate membrane fusion. 293T cells transfected with VZV-gB, gE, gH, gL, and T7 polymerase and 293T cells transfected with MAG and MPRci, as well as with firefly luciferase and Renilla luciferase, were cocultured for 18 h. (D) MAG mediates membrane fusion by associating with HSV-1 glycoproteins. CHO cells transfected with HSV-gB, gD, gH, gL, and T7 polymerase and CHO cells transfected with MAG and firefly luciferase and Renilla luciferase were cocultured for 18 h. (E) Requirement of VZV-gB, gH, and gL for MAG-mediated membrane fusion. 293T cells cotransfected with various combinations of VZV-gB, gE, gH, or gL, as well as T7 polymerase and Renilla luciferase, and 293T cells transfected with MAG, IDE, and firefly luciferase and were cocultured for 18 h. Combinations of glycoproteins used for effector cells were shown. Mock-transfectants were used as control (Cont.). Relative firefly luciferase activities are presented as means ± SD of at least triplicates. Statistical significance (P-value) was shown in the figure. Data are representative of at least three independent experiments.
Fig. 4.
Fig. 4.
MAG enhances VZV infection. (A) VZV infection of MAG-expressing cells. MAG- or mock- (Control) transfected OL cells were infected with VZV-GFP at 0.3 MOI. Expressions of GFP in VZV-infected cells were shown (Upper). Proportions of GFP-expressing cells in mock-infected cells and VZV-GFP-infected cells (VZV-GFP) were analyzed by flow cytometry (Middle and Lower). (B) MAG- (closed circle) or mock- (control, open circle) transfected OL cells were infected with VZV-GFP at the indicated MOI and the proportions of infected cells were calculated. (C) MAG-mediated VZV infection of MRC-5 cells. MAG and TurboRFP were transfected into MRC-5 cells using pMxs-IRES-TurboRFP expression vector, and the transfectants were infected with VZV-GFP. Expressions of TurboRFP (Upper) and GFP (Lower) were analyzed by fluorescence microscopy. (D) Inhibition of VZV infection of MAG-expressing cells by anti-MAG mAb. MAG-expressing OL cells were infected with VZV-GFP at 0.1 MOI in the presence of anti-MAG mAb (closed circle) or isotype-matched control mAb (open circle). Proportions of GFP-positive cells were determined by flow cytometry and the percentage inhibition of VZV infection by mAbs was calculated. All data are presented as means ± SD of duplicates. Data are representative of at least three independent experiments.
Fig. 5.
Fig. 5.
MAG enhances HSV-1 infection. (A) HSV-1 infection of MAG-expressing cells. MAG- or control-transfected HL-60 cells were infected with HSV-GFP at 10 MOI. Expressions of GFP in HSV-1-infected cells were shown (Upper). (B) MAG- (closed circle) or mock- (open circle) transfectants were infected with HSV-GFP at the indicated MOI and the proportions of infected cells were calculated. Data are presented as means ± SD of triplicates. (C) Inhibition of HSV-1 infection of MAG-expressing cells by anti-MAG mAb. MAG-expressing cells were infected with HSV-GFP at 10 MOI in the presence of anti-MAG mAb (closed circle) or isotype-matched control mAb (open circle). The percentage inhibition of HSV-1 infection by mAbs was calculated. Data are presented as means ± SD of triplicates. Data are representative of at least three independent experiments.

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