Limits...
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

In-cell infection occurs in normal ECs. (A) Percentage kinetics of cell-in-cell structure formation between NEPC1-Bmi1 cells and GFP-Akata or AK31 cells. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata or AK31 cells at a ratio of 1:10. The frequencies of cell-in-cell structures were counted at the indicated times. (B) Representative images of EBV activation in an internalized GFP-Akata cells within an NEPC1-Bmi1 cell. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata cells at a ratio of 1:10 for 24 h, followed by fixation and DAPI staining. GFP fluorescence was distributed in internalized GFP-Akata cells. (C) Representative images of the appearance of GFP fluorescence in an NEPC1-Bmi1 cell with an internalized GFP+ GFP-Akata cell. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata cells at a ratio of 1:10 for 48 h, followed by fixation and DAPI staining. GFP fluorescence was distributed in both internalized GFP-Akata cells and NEPC1-Bmi1 cells. (D) Co-localization of GFP fluorescence with LMP2A (upper panel) or EBNA1 (lower panel) in NEPC1-Bmi1 cells. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata cells at a ratio of 1:10 for 24 h, followed by LMP2A or EBNA1 immunofluorescence staining and subsequent DAPI staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493273&req=5

fig4: In-cell infection occurs in normal ECs. (A) Percentage kinetics of cell-in-cell structure formation between NEPC1-Bmi1 cells and GFP-Akata or AK31 cells. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata or AK31 cells at a ratio of 1:10. The frequencies of cell-in-cell structures were counted at the indicated times. (B) Representative images of EBV activation in an internalized GFP-Akata cells within an NEPC1-Bmi1 cell. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata cells at a ratio of 1:10 for 24 h, followed by fixation and DAPI staining. GFP fluorescence was distributed in internalized GFP-Akata cells. (C) Representative images of the appearance of GFP fluorescence in an NEPC1-Bmi1 cell with an internalized GFP+ GFP-Akata cell. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata cells at a ratio of 1:10 for 48 h, followed by fixation and DAPI staining. GFP fluorescence was distributed in both internalized GFP-Akata cells and NEPC1-Bmi1 cells. (D) Co-localization of GFP fluorescence with LMP2A (upper panel) or EBNA1 (lower panel) in NEPC1-Bmi1 cells. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata cells at a ratio of 1:10 for 24 h, followed by LMP2A or EBNA1 immunofluorescence staining and subsequent DAPI staining.

Mentions: We have demonstrated in vitro that EBV-loaded GFP-Akata B cells could invade NPC cell line CNE-2, leading to the transmission of EBV to host CNE-2 cells. However, tumor cells are not equivalent to normal ECs. Therefore, we investigated the occurrence of in-cell infection in normal ECs by using an immortalized epithelial cell line NEPC1-Bmi153 as the host cell. NEPC1-Bmi1 cells formed heterotypic cell-in-cell structures with GFP-Akata cells in a higher frequency than with AK31 cells, which reached 1% for GFP-Akata cells and 0.66% for Ak31 cells at 24 h (Figure 4A). However, the percentage of NEPC1-Bmi1 cells with cell-in-cell structures was lower than that of CNE-2 cells. GFP+ GFP-Akata cells inside NEPC1-Bmi1 cells were detected 24 h after co-culture (Figure 4B). At 48 h, green fluorescence was observed in NEPC1-Bmi1 cells with cell-in-cell structures (Figure 4C). This indicates that NEPC1-Bmi1 cells can be infected by EBV through cell-in-cell pathway. EBV-derived LMP2A and EBNA1 were co-localized with the GFP protein (Figure 4D), which further verified the successful infection of NEPC1-Bmi1 cells through cell-in-cell pathway. We compared the abilities of C-EBV and A-EBV to infect NEPC1-Bmi1 cells. Similar to CNE-2 cells, C-EBV infected NEPC1-Bmi1 cells at a higher frequency than A-EBV (Figure 3B). These results demonstrate that like tumor-derived ECs, normal ECs can also undergo in-cell infection by EBV-infected B cells.


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)

In-cell infection occurs in normal ECs. (A) Percentage kinetics of cell-in-cell structure formation between NEPC1-Bmi1 cells and GFP-Akata or AK31 cells. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata or AK31 cells at a ratio of 1:10. The frequencies of cell-in-cell structures were counted at the indicated times. (B) Representative images of EBV activation in an internalized GFP-Akata cells within an NEPC1-Bmi1 cell. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata cells at a ratio of 1:10 for 24 h, followed by fixation and DAPI staining. GFP fluorescence was distributed in internalized GFP-Akata cells. (C) Representative images of the appearance of GFP fluorescence in an NEPC1-Bmi1 cell with an internalized GFP+ GFP-Akata cell. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata cells at a ratio of 1:10 for 48 h, followed by fixation and DAPI staining. GFP fluorescence was distributed in both internalized GFP-Akata cells and NEPC1-Bmi1 cells. (D) Co-localization of GFP fluorescence with LMP2A (upper panel) or EBNA1 (lower panel) in NEPC1-Bmi1 cells. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata cells at a ratio of 1:10 for 24 h, followed by LMP2A or EBNA1 immunofluorescence staining and subsequent DAPI staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493273&req=5

fig4: In-cell infection occurs in normal ECs. (A) Percentage kinetics of cell-in-cell structure formation between NEPC1-Bmi1 cells and GFP-Akata or AK31 cells. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata or AK31 cells at a ratio of 1:10. The frequencies of cell-in-cell structures were counted at the indicated times. (B) Representative images of EBV activation in an internalized GFP-Akata cells within an NEPC1-Bmi1 cell. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata cells at a ratio of 1:10 for 24 h, followed by fixation and DAPI staining. GFP fluorescence was distributed in internalized GFP-Akata cells. (C) Representative images of the appearance of GFP fluorescence in an NEPC1-Bmi1 cell with an internalized GFP+ GFP-Akata cell. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata cells at a ratio of 1:10 for 48 h, followed by fixation and DAPI staining. GFP fluorescence was distributed in both internalized GFP-Akata cells and NEPC1-Bmi1 cells. (D) Co-localization of GFP fluorescence with LMP2A (upper panel) or EBNA1 (lower panel) in NEPC1-Bmi1 cells. The NEPC1-Bmi1 cells were co-cultured with GFP-Akata cells at a ratio of 1:10 for 24 h, followed by LMP2A or EBNA1 immunofluorescence staining and subsequent DAPI staining.
Mentions: We have demonstrated in vitro that EBV-loaded GFP-Akata B cells could invade NPC cell line CNE-2, leading to the transmission of EBV to host CNE-2 cells. However, tumor cells are not equivalent to normal ECs. Therefore, we investigated the occurrence of in-cell infection in normal ECs by using an immortalized epithelial cell line NEPC1-Bmi153 as the host cell. NEPC1-Bmi1 cells formed heterotypic cell-in-cell structures with GFP-Akata cells in a higher frequency than with AK31 cells, which reached 1% for GFP-Akata cells and 0.66% for Ak31 cells at 24 h (Figure 4A). However, the percentage of NEPC1-Bmi1 cells with cell-in-cell structures was lower than that of CNE-2 cells. GFP+ GFP-Akata cells inside NEPC1-Bmi1 cells were detected 24 h after co-culture (Figure 4B). At 48 h, green fluorescence was observed in NEPC1-Bmi1 cells with cell-in-cell structures (Figure 4C). This indicates that NEPC1-Bmi1 cells can be infected by EBV through cell-in-cell pathway. EBV-derived LMP2A and EBNA1 were co-localized with the GFP protein (Figure 4D), which further verified the successful infection of NEPC1-Bmi1 cells through cell-in-cell pathway. We compared the abilities of C-EBV and A-EBV to infect NEPC1-Bmi1 cells. Similar to CNE-2 cells, C-EBV infected NEPC1-Bmi1 cells at a higher frequency than A-EBV (Figure 3B). These results demonstrate that like tumor-derived ECs, normal ECs can also undergo in-cell infection by EBV-infected B cells.

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