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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

Autonomous activation of EBV inside CNE-2 cells depends on the PI3K/AKT signaling pathway. (A) GFP-Akata cells were co-cultured with CNE-2 cells for 12 h and the phosphorylation of AKT inside GFP-Akata cells was detected by immunofluorescence staining using anti-phospho-AKT antibody followed by DAPI staining. The images were captured under confocal laser scanning microscope. (B) Proportional kinetics of phospho-AKT+ GFP-Akata and GFP+ CNE-2 cells within cell-in-cell structures. GFP-Akata cells were co-cultured with CNE-2 cells. The percentages of phospho-AKT+ GFP-Akata and GFP+CNE-2 cells were analyzed by confocal laser scanning microscopy at the indicated times. The experiments were performed three times independently. (C) The PI3K signaling inhibitor Wortmannin was added to the culture medium after cell-in-cell structure formation (2 or 4 h after co-culture of GFP-Akata and CNE-2 cells). The percentages of phospho-AKT+ GFP-Akata cells (left) and GFP+ GFP-Akata cells (right) within cell-in-cell structures were determined by fluorescence microscopy 24 h after the treatment.
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fig5: Autonomous activation of EBV inside CNE-2 cells depends on the PI3K/AKT signaling pathway. (A) GFP-Akata cells were co-cultured with CNE-2 cells for 12 h and the phosphorylation of AKT inside GFP-Akata cells was detected by immunofluorescence staining using anti-phospho-AKT antibody followed by DAPI staining. The images were captured under confocal laser scanning microscope. (B) Proportional kinetics of phospho-AKT+ GFP-Akata and GFP+ CNE-2 cells within cell-in-cell structures. GFP-Akata cells were co-cultured with CNE-2 cells. The percentages of phospho-AKT+ GFP-Akata and GFP+CNE-2 cells were analyzed by confocal laser scanning microscopy at the indicated times. The experiments were performed three times independently. (C) The PI3K signaling inhibitor Wortmannin was added to the culture medium after cell-in-cell structure formation (2 or 4 h after co-culture of GFP-Akata and CNE-2 cells). The percentages of phospho-AKT+ GFP-Akata cells (left) and GFP+ GFP-Akata cells (right) within cell-in-cell structures were determined by fluorescence microscopy 24 h after the treatment.

Mentions: Activation of EBV in infected B cells by anti-IgG treatment has been well documented49. B-cell receptor (BCR) ligation triggers the activation of the Src tyrosine kinase Lyn and the downstream PI3K/AKT signaling pathway. This activates the transcription of BZLF1, which is a switch for EBV activation from latent status. In turn, BZLF1-encoding protein ZEBRA initiates the replication of virus54. We found that EBV in GFP-Akata cells was activated autonomously inside host CNE-2 cells in the absence of extrinsic stimuli. It is evident that the PI3K/AKT pathway is key to EBV activation in both cell-free and cell-to-cell contact infections50,55. We then asked whether the PI3K/AKT signaling pathway was turned on during the autonomous EBV activation after cell-in-cell structure formation. We detected the phosphorylation of AKTSer473 in GFP-Akata cells with cell-in-cell structures. By 12 h, the GFP fluorescence was co-localized with phosphorylated AKTSer473 in GFP-Akata cells, but was not detected in CNE-2 cells (Figure 5A). Similarly, no phosphorylation of AKT was observed in AK31 cells when they entered CNE-2 cells during the same observation time (Supplementary information, Figure S4). Our results were in accordance with the report55 that EBV immediate-early protein BRLF1 could induce the activation of PI3K/AKT and the subsequent viral activation. This suggests the involvement of the PI3K/AKT signaling in EBV activation within GFP-Akata cells during in-cell infection. When the co-culture continued, the level of phosphorylated AKT increased in GFP+ GFP-Akata cells inside CNE-2 cells (Figure 5B). To further demonstrate the role of the PI3K/AKT signaling in EBV activation, the broad-spectrum PI3K signaling inhibitor, Wortmannin, was added after GFP-Akata cells were co-cultured with CNE-2 cells for 2 or 4 h. After another 24 h co-culture, GFP signal and AKT phosphorylation were measured in cell-in-cell structures. As shown in Figure 5C, Wortmannin dramatically inhibited the phosphorylation of AKT inside GFP-Akata cells (Figure 5C, left). In parallel, GFP expression in GFP-Akata cells inside CNE-2 cells also decreased dramatically (Figure 5C, right). We obtained the similar results when we used another PI3K inhibitor LY296002 (data not shown). These results suggest that the PI3K/AKT signaling is involved in EBV activation within B cells after cell-in-cell structure formation.


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)

Autonomous activation of EBV inside CNE-2 cells depends on the PI3K/AKT signaling pathway. (A) GFP-Akata cells were co-cultured with CNE-2 cells for 12 h and the phosphorylation of AKT inside GFP-Akata cells was detected by immunofluorescence staining using anti-phospho-AKT antibody followed by DAPI staining. The images were captured under confocal laser scanning microscope. (B) Proportional kinetics of phospho-AKT+ GFP-Akata and GFP+ CNE-2 cells within cell-in-cell structures. GFP-Akata cells were co-cultured with CNE-2 cells. The percentages of phospho-AKT+ GFP-Akata and GFP+CNE-2 cells were analyzed by confocal laser scanning microscopy at the indicated times. The experiments were performed three times independently. (C) The PI3K signaling inhibitor Wortmannin was added to the culture medium after cell-in-cell structure formation (2 or 4 h after co-culture of GFP-Akata and CNE-2 cells). The percentages of phospho-AKT+ GFP-Akata cells (left) and GFP+ GFP-Akata cells (right) within cell-in-cell structures were determined by fluorescence microscopy 24 h after the treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Autonomous activation of EBV inside CNE-2 cells depends on the PI3K/AKT signaling pathway. (A) GFP-Akata cells were co-cultured with CNE-2 cells for 12 h and the phosphorylation of AKT inside GFP-Akata cells was detected by immunofluorescence staining using anti-phospho-AKT antibody followed by DAPI staining. The images were captured under confocal laser scanning microscope. (B) Proportional kinetics of phospho-AKT+ GFP-Akata and GFP+ CNE-2 cells within cell-in-cell structures. GFP-Akata cells were co-cultured with CNE-2 cells. The percentages of phospho-AKT+ GFP-Akata and GFP+CNE-2 cells were analyzed by confocal laser scanning microscopy at the indicated times. The experiments were performed three times independently. (C) The PI3K signaling inhibitor Wortmannin was added to the culture medium after cell-in-cell structure formation (2 or 4 h after co-culture of GFP-Akata and CNE-2 cells). The percentages of phospho-AKT+ GFP-Akata cells (left) and GFP+ GFP-Akata cells (right) within cell-in-cell structures were determined by fluorescence microscopy 24 h after the treatment.
Mentions: Activation of EBV in infected B cells by anti-IgG treatment has been well documented49. B-cell receptor (BCR) ligation triggers the activation of the Src tyrosine kinase Lyn and the downstream PI3K/AKT signaling pathway. This activates the transcription of BZLF1, which is a switch for EBV activation from latent status. In turn, BZLF1-encoding protein ZEBRA initiates the replication of virus54. We found that EBV in GFP-Akata cells was activated autonomously inside host CNE-2 cells in the absence of extrinsic stimuli. It is evident that the PI3K/AKT pathway is key to EBV activation in both cell-free and cell-to-cell contact infections50,55. We then asked whether the PI3K/AKT signaling pathway was turned on during the autonomous EBV activation after cell-in-cell structure formation. We detected the phosphorylation of AKTSer473 in GFP-Akata cells with cell-in-cell structures. By 12 h, the GFP fluorescence was co-localized with phosphorylated AKTSer473 in GFP-Akata cells, but was not detected in CNE-2 cells (Figure 5A). Similarly, no phosphorylation of AKT was observed in AK31 cells when they entered CNE-2 cells during the same observation time (Supplementary information, Figure S4). Our results were in accordance with the report55 that EBV immediate-early protein BRLF1 could induce the activation of PI3K/AKT and the subsequent viral activation. This suggests the involvement of the PI3K/AKT signaling in EBV activation within GFP-Akata cells during in-cell infection. When the co-culture continued, the level of phosphorylated AKT increased in GFP+ GFP-Akata cells inside CNE-2 cells (Figure 5B). To further demonstrate the role of the PI3K/AKT signaling in EBV activation, the broad-spectrum PI3K signaling inhibitor, Wortmannin, was added after GFP-Akata cells were co-cultured with CNE-2 cells for 2 or 4 h. After another 24 h co-culture, GFP signal and AKT phosphorylation were measured in cell-in-cell structures. As shown in Figure 5C, Wortmannin dramatically inhibited the phosphorylation of AKT inside GFP-Akata cells (Figure 5C, left). In parallel, GFP expression in GFP-Akata cells inside CNE-2 cells also decreased dramatically (Figure 5C, right). We obtained the similar results when we used another PI3K inhibitor LY296002 (data not shown). These results suggest that the PI3K/AKT signaling is involved in EBV activation within B cells after cell-in-cell structure formation.

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