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In-cell infection: a novel pathway for Epstein-Barr virus infection mediated by cell-in-cell structures.

Ni C, Chen Y, Zeng M, Pei R, Du Y, Tang L, Wang M, Hu Y, Zhu H, He M, Wei X, Wang S, Ning X, Wang M, Wang J, Ma L, Chen X, Sun Q, Tang H, Wang Y, Wang X - Cell Res. (2015)

Bottom Line: Epithelial CNE-2 cells were invaded by EBV-infected Akata B cells to form cell-in-cell structures in vitro.Such unique cellular structures could be readily observed in the specimens of nasopharyngeal carcinoma.Importantly, the formation of cell-in-cell structures led to the autonomous activation of EBV within Akata cells and subsequent viral transmission to CNE-2 cells, as evidenced by the expression of viral genes and the presence of virion particles in CNE-2 cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Sciences, Chinese PLA General Hospital and School of Bioscience and Bioengineering, South China University of Technology, Key Laboratory of Normal aging and Geriatric & the State Key Laboratory of Kidney, Beijing 100853 & the Provincial Key Laboratory of Biotechnology, Guangdong 510006, China.

ABSTRACT
Epstein-Barr virus (EBV) can infect both susceptible B lymphocytes and non-susceptible epithelial cells (ECs). Viral tropism analyses have revealed two intriguing means of EBV infection, either by a receptor-mediated infection of B cells or by a cell-to-cell contact-mediated infection of non-susceptible ECs. Herein, we report a novel "in-cell infection" mechanism for EBV infection of non-susceptible ECs through the formation of cell-in-cell structures. Epithelial CNE-2 cells were invaded by EBV-infected Akata B cells to form cell-in-cell structures in vitro. Such unique cellular structures could be readily observed in the specimens of nasopharyngeal carcinoma. Importantly, the formation of cell-in-cell structures led to the autonomous activation of EBV within Akata cells and subsequent viral transmission to CNE-2 cells, as evidenced by the expression of viral genes and the presence of virion particles in CNE-2 cells. Significantly, EBV generated from in-cell infected ECs displayed altered tropism with higher infection efficacy to both B cells and ECs. In addition to CNE-2 tumor cells, cell-in-cell structure formation could also mediate EBV infection of NPEC1-Bmi1 cells, an immortalized nasopharyngeal epithelial cell line. Furthermore, efficient infection by this mechanism involved the activation of the PI3K/AKT signaling pathway. Thus, our study identified "in-cell infection" as a novel mechanism for EBV infection. Given the diversity of virus-infected cells and the prevalence of cell-in-cell structures during chronic infection, we speculate that "in-cell infection" is likely a general mechanism for EBV and other viruses to infect non-susceptible ECs.

No MeSH data available.


Related in: MedlinePlus

EBV-carrying GFP-Akaka cells transmit virus to CNE-2 cells through cell-in-cell interaction. (A) Time tracking observation of EBV activation in GFP-Akata cells within CNE-2 cells. CNE-2 cells (pre-stained with CellTracker Red dye) were co-cultured with GFP-Akata cells at a ratio of 1:10 and observed using LSM 710 confocal microscope. It was notable that internalized GFP-Akata cells became green (GFP-positive), which indicated EBV activation, at time 07:15:00. Time was indicated as hour:minute:second. Scale bar,10 μm. (B) The fate of individual GFP-Akata cell was indicated, including the appearance of GFP (green circle), undergoing cell-in-cell death (black circle) or escaping from CNE-2 cells (red triangle). Data analysis was performed for 12 h with an 1 h interval. (C) Expression of ZEBRA (red) in an EBV-activated GFP-Akata cell (green) inside a CNE-2 cell (indicated by DAPI) as determined by immunofluorescence staining. (D) Time tracking analysis of GFP diffusion into CNE-2 cells. The CNE-2 cells were co-cultured with GFP-Akata cells at a ratio of 1:5 and observed using DMI6000B fluorescence microscope. The target CNE-2 cells became GFP positive at time 39:50:00, which was the indicator of EBV infection. Time was indicated as hour:minute:second. Scale bar, 10 μm. (E) Distribution of EBER (red) and GFP (green) in cell-in-cell structures. After incubation of GFP-Akata cells with CNE-2 cells for 12 h, the free GFP-Akata cells were removed by washing with PBS. The sorted CNE-2 cells with cell-in-cell structures were hybridized with the probe to EBER for ISH. (F) PCR analysis of the indicated viral mRNAs in the sorted CNE-2 cells with cell-in-cell structures. GFP-Akata cells served as positive control while non-treated CNE-2 cells served as negative control. (G) TEM observation of EBV virions (yellow circle) in in-cell infected CNE-2 cells. The in-cell infected CNE-2 cells were obtained by FACS sorting after co-culturing with GFP-Akata cells at a ratio of 1: 10 and maintained with G418-containing culture medium for 2 weeks. (H) A summary of cell-in-cell- and cell-to-cell-mediated EBV transmission. CNE-2 cells were co-cultured with GFP-Akata cells (with or without anti-human IgG treatment) for 24 h. The frequencies of GFP-positive CNE-2 cells freely or with cell-in-cell structures were analyzed by fluorescence microscopy.
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fig2: EBV-carrying GFP-Akaka cells transmit virus to CNE-2 cells through cell-in-cell interaction. (A) Time tracking observation of EBV activation in GFP-Akata cells within CNE-2 cells. CNE-2 cells (pre-stained with CellTracker Red dye) were co-cultured with GFP-Akata cells at a ratio of 1:10 and observed using LSM 710 confocal microscope. It was notable that internalized GFP-Akata cells became green (GFP-positive), which indicated EBV activation, at time 07:15:00. Time was indicated as hour:minute:second. Scale bar,10 μm. (B) The fate of individual GFP-Akata cell was indicated, including the appearance of GFP (green circle), undergoing cell-in-cell death (black circle) or escaping from CNE-2 cells (red triangle). Data analysis was performed for 12 h with an 1 h interval. (C) Expression of ZEBRA (red) in an EBV-activated GFP-Akata cell (green) inside a CNE-2 cell (indicated by DAPI) as determined by immunofluorescence staining. (D) Time tracking analysis of GFP diffusion into CNE-2 cells. The CNE-2 cells were co-cultured with GFP-Akata cells at a ratio of 1:5 and observed using DMI6000B fluorescence microscope. The target CNE-2 cells became GFP positive at time 39:50:00, which was the indicator of EBV infection. Time was indicated as hour:minute:second. Scale bar, 10 μm. (E) Distribution of EBER (red) and GFP (green) in cell-in-cell structures. After incubation of GFP-Akata cells with CNE-2 cells for 12 h, the free GFP-Akata cells were removed by washing with PBS. The sorted CNE-2 cells with cell-in-cell structures were hybridized with the probe to EBER for ISH. (F) PCR analysis of the indicated viral mRNAs in the sorted CNE-2 cells with cell-in-cell structures. GFP-Akata cells served as positive control while non-treated CNE-2 cells served as negative control. (G) TEM observation of EBV virions (yellow circle) in in-cell infected CNE-2 cells. The in-cell infected CNE-2 cells were obtained by FACS sorting after co-culturing with GFP-Akata cells at a ratio of 1: 10 and maintained with G418-containing culture medium for 2 weeks. (H) A summary of cell-in-cell- and cell-to-cell-mediated EBV transmission. CNE-2 cells were co-cultured with GFP-Akata cells (with or without anti-human IgG treatment) for 24 h. The frequencies of GFP-positive CNE-2 cells freely or with cell-in-cell structures were analyzed by fluorescence microscopy.

Mentions: To directly determine whether EBV-infected B cells could transmit EBV to uninfected ECs by invading into ECs, we co-cultured EBV-negative CNE-2 cells, a human NPC-derived EC cell line without CD21/CR2 receptors48, with BL-derived EBV-eGFP Akata cells. EBV-eGFP Akata cells were derived from EBV− AK31 cell line that was latently infected with recombinant EBV containing an eGFP gene and a neomycin-resistant gene. eGFP gene was inserted into the viral BXLF1 open reading frame to monitor the activation of EBV and the infection of ECs47. This cell line was referred to as GFP-Akata cells afterward. Consistent with the observations in clinical samples described above, both GFP-Akata cells and parent AK31 cells formed heterotypic cell-in-cell structures with CNE-2 cells after the co-culture in vitro. Remarkably, GFP-Akata cells formed cell-in-cell structures in higher frequency compared with uninfected parent AK31 cells in parallel (Supplementary information, Figure S1A). To determine whether GFP-Akata cells could transfer EBV to CNE-2 cells through the cell-in-cell mechanism, we used time-lapse microscope to track the invading processes of GFP-Akata cells into CNE-2 cells. We observed the gradual increase of green fluorescence in the internalized GFP-Akata cell 6 h after the initiation of co-culture, indicating the activation of EBV. The GFP intensity and the percentage of internalized GFP+ Akata cells continued to increase until 10 h (Figure 2A). However, free GFP-Akata cells maintained the similar GFP positivity during co-culture (data not shown). Internalized GFP-Akata cells appeared intact during the observation period (Figure 2A). We individually tracked 135 cell-in-cell structures with GFP− GFP-Akata cells at the beginning of the observation for 12 h. Fifteen internalized cells turned green over the interval (Figure 2B). Among them, three went out of host cells intactly (Figure 2B and Supplementary information, Figure S1C). Most internalized GFP-Akata cells displayed characteristics of entotic cell death (lysotracker positive and caspase-3 negative; Supplementary information, Figure S1B). No green fluorescence was observed in CNE-2 cells without cell-in-cell structures.


In-cell infection: a novel pathway for Epstein-Barr virus infection mediated by cell-in-cell structures.

Ni C, Chen Y, Zeng M, Pei R, Du Y, Tang L, Wang M, Hu Y, Zhu H, He M, Wei X, Wang S, Ning X, Wang M, Wang J, Ma L, Chen X, Sun Q, Tang H, Wang Y, Wang X - Cell Res. (2015)

EBV-carrying GFP-Akaka cells transmit virus to CNE-2 cells through cell-in-cell interaction. (A) Time tracking observation of EBV activation in GFP-Akata cells within CNE-2 cells. CNE-2 cells (pre-stained with CellTracker Red dye) were co-cultured with GFP-Akata cells at a ratio of 1:10 and observed using LSM 710 confocal microscope. It was notable that internalized GFP-Akata cells became green (GFP-positive), which indicated EBV activation, at time 07:15:00. Time was indicated as hour:minute:second. Scale bar,10 μm. (B) The fate of individual GFP-Akata cell was indicated, including the appearance of GFP (green circle), undergoing cell-in-cell death (black circle) or escaping from CNE-2 cells (red triangle). Data analysis was performed for 12 h with an 1 h interval. (C) Expression of ZEBRA (red) in an EBV-activated GFP-Akata cell (green) inside a CNE-2 cell (indicated by DAPI) as determined by immunofluorescence staining. (D) Time tracking analysis of GFP diffusion into CNE-2 cells. The CNE-2 cells were co-cultured with GFP-Akata cells at a ratio of 1:5 and observed using DMI6000B fluorescence microscope. The target CNE-2 cells became GFP positive at time 39:50:00, which was the indicator of EBV infection. Time was indicated as hour:minute:second. Scale bar, 10 μm. (E) Distribution of EBER (red) and GFP (green) in cell-in-cell structures. After incubation of GFP-Akata cells with CNE-2 cells for 12 h, the free GFP-Akata cells were removed by washing with PBS. The sorted CNE-2 cells with cell-in-cell structures were hybridized with the probe to EBER for ISH. (F) PCR analysis of the indicated viral mRNAs in the sorted CNE-2 cells with cell-in-cell structures. GFP-Akata cells served as positive control while non-treated CNE-2 cells served as negative control. (G) TEM observation of EBV virions (yellow circle) in in-cell infected CNE-2 cells. The in-cell infected CNE-2 cells were obtained by FACS sorting after co-culturing with GFP-Akata cells at a ratio of 1: 10 and maintained with G418-containing culture medium for 2 weeks. (H) A summary of cell-in-cell- and cell-to-cell-mediated EBV transmission. CNE-2 cells were co-cultured with GFP-Akata cells (with or without anti-human IgG treatment) for 24 h. The frequencies of GFP-positive CNE-2 cells freely or with cell-in-cell structures were analyzed by fluorescence microscopy.
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fig2: EBV-carrying GFP-Akaka cells transmit virus to CNE-2 cells through cell-in-cell interaction. (A) Time tracking observation of EBV activation in GFP-Akata cells within CNE-2 cells. CNE-2 cells (pre-stained with CellTracker Red dye) were co-cultured with GFP-Akata cells at a ratio of 1:10 and observed using LSM 710 confocal microscope. It was notable that internalized GFP-Akata cells became green (GFP-positive), which indicated EBV activation, at time 07:15:00. Time was indicated as hour:minute:second. Scale bar,10 μm. (B) The fate of individual GFP-Akata cell was indicated, including the appearance of GFP (green circle), undergoing cell-in-cell death (black circle) or escaping from CNE-2 cells (red triangle). Data analysis was performed for 12 h with an 1 h interval. (C) Expression of ZEBRA (red) in an EBV-activated GFP-Akata cell (green) inside a CNE-2 cell (indicated by DAPI) as determined by immunofluorescence staining. (D) Time tracking analysis of GFP diffusion into CNE-2 cells. The CNE-2 cells were co-cultured with GFP-Akata cells at a ratio of 1:5 and observed using DMI6000B fluorescence microscope. The target CNE-2 cells became GFP positive at time 39:50:00, which was the indicator of EBV infection. Time was indicated as hour:minute:second. Scale bar, 10 μm. (E) Distribution of EBER (red) and GFP (green) in cell-in-cell structures. After incubation of GFP-Akata cells with CNE-2 cells for 12 h, the free GFP-Akata cells were removed by washing with PBS. The sorted CNE-2 cells with cell-in-cell structures were hybridized with the probe to EBER for ISH. (F) PCR analysis of the indicated viral mRNAs in the sorted CNE-2 cells with cell-in-cell structures. GFP-Akata cells served as positive control while non-treated CNE-2 cells served as negative control. (G) TEM observation of EBV virions (yellow circle) in in-cell infected CNE-2 cells. The in-cell infected CNE-2 cells were obtained by FACS sorting after co-culturing with GFP-Akata cells at a ratio of 1: 10 and maintained with G418-containing culture medium for 2 weeks. (H) A summary of cell-in-cell- and cell-to-cell-mediated EBV transmission. CNE-2 cells were co-cultured with GFP-Akata cells (with or without anti-human IgG treatment) for 24 h. The frequencies of GFP-positive CNE-2 cells freely or with cell-in-cell structures were analyzed by fluorescence microscopy.
Mentions: To directly determine whether EBV-infected B cells could transmit EBV to uninfected ECs by invading into ECs, we co-cultured EBV-negative CNE-2 cells, a human NPC-derived EC cell line without CD21/CR2 receptors48, with BL-derived EBV-eGFP Akata cells. EBV-eGFP Akata cells were derived from EBV− AK31 cell line that was latently infected with recombinant EBV containing an eGFP gene and a neomycin-resistant gene. eGFP gene was inserted into the viral BXLF1 open reading frame to monitor the activation of EBV and the infection of ECs47. This cell line was referred to as GFP-Akata cells afterward. Consistent with the observations in clinical samples described above, both GFP-Akata cells and parent AK31 cells formed heterotypic cell-in-cell structures with CNE-2 cells after the co-culture in vitro. Remarkably, GFP-Akata cells formed cell-in-cell structures in higher frequency compared with uninfected parent AK31 cells in parallel (Supplementary information, Figure S1A). To determine whether GFP-Akata cells could transfer EBV to CNE-2 cells through the cell-in-cell mechanism, we used time-lapse microscope to track the invading processes of GFP-Akata cells into CNE-2 cells. We observed the gradual increase of green fluorescence in the internalized GFP-Akata cell 6 h after the initiation of co-culture, indicating the activation of EBV. The GFP intensity and the percentage of internalized GFP+ Akata cells continued to increase until 10 h (Figure 2A). However, free GFP-Akata cells maintained the similar GFP positivity during co-culture (data not shown). Internalized GFP-Akata cells appeared intact during the observation period (Figure 2A). We individually tracked 135 cell-in-cell structures with GFP− GFP-Akata cells at the beginning of the observation for 12 h. Fifteen internalized cells turned green over the interval (Figure 2B). Among them, three went out of host cells intactly (Figure 2B and Supplementary information, Figure S1C). Most internalized GFP-Akata cells displayed characteristics of entotic cell death (lysotracker positive and caspase-3 negative; Supplementary information, Figure S1B). No green fluorescence was observed in CNE-2 cells without cell-in-cell structures.

Bottom Line: Epithelial CNE-2 cells were invaded by EBV-infected Akata B cells to form cell-in-cell structures in vitro.Such unique cellular structures could be readily observed in the specimens of nasopharyngeal carcinoma.Importantly, the formation of cell-in-cell structures led to the autonomous activation of EBV within Akata cells and subsequent viral transmission to CNE-2 cells, as evidenced by the expression of viral genes and the presence of virion particles in CNE-2 cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Sciences, Chinese PLA General Hospital and School of Bioscience and Bioengineering, South China University of Technology, Key Laboratory of Normal aging and Geriatric & the State Key Laboratory of Kidney, Beijing 100853 & the Provincial Key Laboratory of Biotechnology, Guangdong 510006, China.

ABSTRACT
Epstein-Barr virus (EBV) can infect both susceptible B lymphocytes and non-susceptible epithelial cells (ECs). Viral tropism analyses have revealed two intriguing means of EBV infection, either by a receptor-mediated infection of B cells or by a cell-to-cell contact-mediated infection of non-susceptible ECs. Herein, we report a novel "in-cell infection" mechanism for EBV infection of non-susceptible ECs through the formation of cell-in-cell structures. Epithelial CNE-2 cells were invaded by EBV-infected Akata B cells to form cell-in-cell structures in vitro. Such unique cellular structures could be readily observed in the specimens of nasopharyngeal carcinoma. Importantly, the formation of cell-in-cell structures led to the autonomous activation of EBV within Akata cells and subsequent viral transmission to CNE-2 cells, as evidenced by the expression of viral genes and the presence of virion particles in CNE-2 cells. Significantly, EBV generated from in-cell infected ECs displayed altered tropism with higher infection efficacy to both B cells and ECs. In addition to CNE-2 tumor cells, cell-in-cell structure formation could also mediate EBV infection of NPEC1-Bmi1 cells, an immortalized nasopharyngeal epithelial cell line. Furthermore, efficient infection by this mechanism involved the activation of the PI3K/AKT signaling pathway. Thus, our study identified "in-cell infection" as a novel mechanism for EBV infection. Given the diversity of virus-infected cells and the prevalence of cell-in-cell structures during chronic infection, we speculate that "in-cell infection" is likely a general mechanism for EBV and other viruses to infect non-susceptible ECs.

No MeSH data available.


Related in: MedlinePlus