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. 2009 Jun 5;388(2):335-43.
doi: 10.1016/j.virol.2009.03.030. Epub 2009 Apr 24.

"V体育ios版" EBV BMRF-2 facilitates cell-to-cell spread of virus within polarized oral epithelial cells

Jianqiao Xiao  1 , Joel M Palefsky, Rossana Herrera, Jennifer Berline, Sharof M Tugizov
Affiliations

EBV BMRF-2 facilitates cell-to-cell spread of virus within polarized oral epithelial cells

Jianqiao Xiao et al. Virology. .

Abstract

We previously reported that the Epstein-Barr virus (EBV) BMRF-2 protein plays an important role in EBV infection of polarized oral epithelial cells by interacting with beta1 and alphav family integrins VSports手机版. Here we show that infection of polarized oral epithelial cells with B27-BMRF-2(low) recombinant virus, expressing a low level of BMRF-2, resulted in significantly smaller plaques compared with infection by parental B95-8 virus. BMRF-2 localized in the trans-Golgi network (TGN) and basolateral sorting vesicles and was transported to the basolateral membranes of polarized epithelial cells. Mutation of the tyrosine- and dileucine-containing basolateral sorting signal, YLLV, in the cytoplasmic domain of BMRF-2 led to the failure of its accumulation in the TGN and its basolateral transport. These data show that BMRF-2 may play an important role in promoting the spread of EBV progeny virions through lateral membranes of oral epithelial cells. .

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V体育官网 - Figures

Fig. 1.
Fig. 1.
Cell-to-cell spread of B95-8 and B27-BMRF-2low virus within the polarized tongue and tonsil epithelial cells. (A) Polarized tongue TNG#1 cells were infected with B95-8 (upper panels) or B27-BMRF-2low virus (lower panel), and after 3 weeks they were fixed and immunostained using anti-gp350/250 antibodies (red). Green in the lower panels indicates GFP expression by the BMRF-2 knockout virus. Yellow in the merged lower panel represents colocalization of GFP and gp350/220. Nuclei were counterstained in blue. (B) Polarized tongue and tonsil cells were infected with B95-8 or B27-BMRF-2low viruses, and after 3 weeks cells were analyzed for virus spread. Cell-to-cell spread of viruses was quantitatively evaluated by counting of EBV-infected cells in B95-8- and B27-BMRF-2low- infected plaques. EBV B95-8-infected plaques were detected by gp350/220-positive cells. B27-BMRF-2low -infected plaques were identified by co-localization of the GFP signal with gp350/220. Results are represented as the average number of EBV-infected cells per plaque. Error bars indicate s.e.m. (n=5). Similar results were obtained in three independent experiments.
Fig. 2
Fig. 2
Analysis of cell-to-cell spread of B27-BMRF-2low virus. Polarized tongue epithelial cells (TNG#1) were infected with B27-BMRF-2low virus from their basolateral membranes, and after 3 weeks, cells were fixed and analyzed for plaque development. (A) Cells were immunostained with rat anti-BMRF-2 antibody (red). Cell nuclei were counterstained in blue. Green in upper panels indicates B27-BMRF-2low virus infected cells. (B) Cells were immunostained with rat anti-BMRF-2 (red) and mouse antigp350/220 (blue) antibodies. Green GFP signal indicates B27-BMRF-2low virus infected cells. White arrow shows BMRF-2–negative, gp350-positive and GFP-positive small plaque. Red arrow shows a BMRF-2–positive, gp350-positive and GFP-negative large plaque.
Fig. 3
Fig. 3
Expression of the wt BMRF-2 and its tyrosine and di-leucine mutants in oral epithelial cells. (A) Site-specific mutagenesis of the BMRF-2 cytoplasmic sorting motif YLLV (in bold) with substitution of tyrosine349 with alanine (BMRF-2Y-A) and the di-leucine350–351 with glycines (BMRF-2 LL-GG. (B) Western blot analysis of wt and mutant BMRF-2 proteins in HSC-BMRF-2wt, HSC-BMRF-2Y-A and HSC-BMRF-2LL-GG cells. Membrane fractions were separated on urea-SDS PAGE gels and transferred to nitrocellulose membranes. BMRF-2 protein was detected using rat anti-BMRF-2 serum.
Fig. 4
Fig. 4
Flow cytometry analysis of cell surface expression of wt and mutant BMRF-2 proteins in non-polarized HSC-3 oral epithelial cells. HSC-BMRF-2wt, HSC-BMRF-2Y-A and HSC-BMRF-2LL-GG cell lines expressing wt BMRF-2 and its tyrosine and dileucine mutants were grown under non-polarizing conditions and dissociated with enzyme-free cell-dissociation buffer. Expression of BMRF-2 on the cell surface was examined by flow cytometry in HSC-BMRF-2wt (left panel), HSC-BMRF-2Y-A (middle panel), and HSC-BMRF-2LL-GG (right panel) cell lines using rat anti-BMRF-2 serum. Surface expression of BMRF-2Y-A and BMRF-2LL-GG mutant proteins were compared with wt BMRF-2 by superimposing the histograms for the HSC-BMRF-2Y-A and HSC-BMRF-2LL-GG cell lines on the histogram for HSC-BMRF-2wt cells. MN, mean fluorescence intensity.
Fig. 5
Fig. 5
Analysis of BMRF-2 protein on the surface of polarized cells using a domain-selective surface labeling assay. HSC-BMRF-2wt, HSC-BMRF-2Y-A and HSC-BMRF-2LL-GG cell lines were grown under polarized conditions and their apical or basolateral surfaces were independently labeled with sulfo-NHS-LC-biotin. Cells extracts were made, biotinylated proteins were precipitated with streptavidin-agarose beads and equal amounts of total protein were separated by urea-SDS PAGE gel electrophoresis. BMRF-2 protein was detected using rat anti-BMRF-2 serum. The amounts of wt and mutant BMRF-2 proteins were measured by determining the mean pixel densities of the protein bands, as indicated in the bar graphs under each protein.
Fig. 6
Fig. 6
Confocal microscopy analysis of basolateral membrane transport of the wt and mutant BMRF-2 proteins. Polarized HSC-BMRF-2wt(A), HSC-BMRF-2Y-A (B) and HSCBMRF- 2LL-GG (C) cell lines were immunostained for pan-cadherin and analyzed by confocal microscopy by x-y horizontal and x-z vertical planes. Nuclei were counterstained in blue. Yellow in the merged panel indicates colocalization of BMRF-2– GFP (green) and pan-cadherin (red) on the basolateral cell surface.
Fig. 7
Fig. 7
Localization of wt and mutant BMRF-2 proteins in the Golgi compartment. Polarized HSC-BMRF-2wt and HSC-BMRF-2Y-A cell lines were fixed and stained with the Golgi marker Rhodamine-Lens Culinaris Agglutinin. BMRF-2 is shown in green fluorescence from a GFP fusion protein, and the Golgi marker is shown in red. Yellow indicates colocalization of BMRF-2 with the Golgi marker.
Fig. 8
Fig. 8
Accumulation of BMRF-2 in the TGN and in the basolateral sorting vesicles of polarized HSC-3 cells. Polarized HSC-BMRF-2wt and HSC-BMRF-2Y-A cell lines were immunostained for TGN46 (A) and AP4μ (B), and colocalization of wt and mutant BMRF-2 proteins with TGN46 and AP-4μ was examined by confocal microscopy. Yellow in the merged panel indicates colocalization of BMRF-2-GFP (green) and TGN46 or AP4μ (red) proteins. Cell nuclei were counterstained in blue.
Fig. 9
Fig. 9
Confocal microscopy analysis of BMRF-2 interaction with β1 integrin in polarized oral epithelial cells. Polarized HSC-BMRF-2wt (A), HSC-BMRF-2Y-A (B) and HSC-BMRF-2LL-GG (C) cells were immunostained for β1 integrin and then examined by confocal microscopy. Images were obtained in both x-y horizontal and x-z vertical planes. Yellow in the merged panel indicates colocalization of BMRF-2–GFP (green) and β integrin (red). Cell nuclei were counterstained in blue.
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