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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 from in-cell infected CNE-2 cells possesses enhanced infection potency and altered tropism. (A) Determination of GFP+ AK31 cells (left) and CNE-2 cells (right) after the infection with A-EBV from GFP-Akata cells (blue line) or C-EBV from iCNE-2 cells (red line) for 24 h by flow cytometry. Cells without EBV treatment served as negative control (black line). (B) Statistical comparison of percentages of CNE-2, A431, MCF-7, PLC/PRF/5, AK31 and NEPC1-Bmi1 cells with GFP expression after 24 h cell-free infection with A-EBV or C-EBV by immunofluorescence microscopy. All experiments were repeated at least three times. *P < 0.05. (C, D) Absorption assay of A-EBV (C) or C-EBV (D) was performed. 2 × 107 DNA copies/ml A-EBV (C) or C-EBV (D) virus was incubated with 2 × 106 AK31 or CNE-2 cells for 2 h on ice. Supernatants were collected and the fresh medium was added to cells for culture. The percentage of GFP+ AK31 or CNE-2 cells was determined by flow cytometry after 12 h. The collected supernatants were added to fresh AK31 and CNE-2 cells to determine the infection rate after 12 h. (E) AK31 or Raji cells were co-cultured with in-cell infected iCNE-2 cells for 24 h. The expression of GFP in AK31 (upper) or Raji (lower) cells was analyzed by flow cytometry. AK31 and Raji cells only (left) served as negative controls.
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fig3: EBV from in-cell infected CNE-2 cells possesses enhanced infection potency and altered tropism. (A) Determination of GFP+ AK31 cells (left) and CNE-2 cells (right) after the infection with A-EBV from GFP-Akata cells (blue line) or C-EBV from iCNE-2 cells (red line) for 24 h by flow cytometry. Cells without EBV treatment served as negative control (black line). (B) Statistical comparison of percentages of CNE-2, A431, MCF-7, PLC/PRF/5, AK31 and NEPC1-Bmi1 cells with GFP expression after 24 h cell-free infection with A-EBV or C-EBV by immunofluorescence microscopy. All experiments were repeated at least three times. *P < 0.05. (C, D) Absorption assay of A-EBV (C) or C-EBV (D) was performed. 2 × 107 DNA copies/ml A-EBV (C) or C-EBV (D) virus was incubated with 2 × 106 AK31 or CNE-2 cells for 2 h on ice. Supernatants were collected and the fresh medium was added to cells for culture. The percentage of GFP+ AK31 or CNE-2 cells was determined by flow cytometry after 12 h. The collected supernatants were added to fresh AK31 and CNE-2 cells to determine the infection rate after 12 h. (E) AK31 or Raji cells were co-cultured with in-cell infected iCNE-2 cells for 24 h. The expression of GFP in AK31 (upper) or Raji (lower) cells was analyzed by flow cytometry. AK31 and Raji cells only (left) served as negative controls.

Mentions: We stimulated GFP-Akata and iCNE-2 cells with anti-human IgG49 and sodium butyrate52, respectively, to initiate the EBV virion production. After stimulation for 3 days, culture supernatants containing EBV virions were collected. To distinguish EBV virions derived from these two types of cells, A-EBV and C-EBV were named for EBVs from GFP-Akata and iCNE-2 cells, respectively. By using the same amount of virions normalized by quantitative PCR, we first performed the cell-free infection assay in susceptible AK31 and non-susceptible CNE-2 cells to compare the infectivity between A-EBV and C-EBV. A-EBV maintained the capacity to infect AK31 cells at the percentage of ∼10% but could not infect CNE-2 cells (Figure 3A left and right, blue line for A-EBV infection). Surprisingly, based on the expression of GFP fluorescence, we found that C-EBV from iCNE-2 cells infected both AK31 (Figure 3A, left, red line, 10.1%) and CNE-2 cells (Figure 3A, right, red line, 14.8%). It is well established that deficiency of EBV receptor such as CD21 molecules on ECs is the main obstacle for their susceptibility to cell-free EBV infection19. However, the extra ability of C-EBV to infect CNE-2 cells suggests that CNE-2 cells infected via in-cell infection are able to assemble virions with an extra capacity to infect non-susceptible ECs. This was confirmed when we further compared the capacity of A-EBV and C-EBV to infect other lymphocytes or EC-derived cell lines (Figure 3B). While A-EBV only infected B cell line AK31, C-EBV infected both lymphocytes (AK31) and EC cell lines (i.e., CNE-2, A431, MCF-7, PLC/PRF/5 and an immortalized epithelial cell line NPEC1-Bmi1)53.


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 from in-cell infected CNE-2 cells possesses enhanced infection potency and altered tropism. (A) Determination of GFP+ AK31 cells (left) and CNE-2 cells (right) after the infection with A-EBV from GFP-Akata cells (blue line) or C-EBV from iCNE-2 cells (red line) for 24 h by flow cytometry. Cells without EBV treatment served as negative control (black line). (B) Statistical comparison of percentages of CNE-2, A431, MCF-7, PLC/PRF/5, AK31 and NEPC1-Bmi1 cells with GFP expression after 24 h cell-free infection with A-EBV or C-EBV by immunofluorescence microscopy. All experiments were repeated at least three times. *P < 0.05. (C, D) Absorption assay of A-EBV (C) or C-EBV (D) was performed. 2 × 107 DNA copies/ml A-EBV (C) or C-EBV (D) virus was incubated with 2 × 106 AK31 or CNE-2 cells for 2 h on ice. Supernatants were collected and the fresh medium was added to cells for culture. The percentage of GFP+ AK31 or CNE-2 cells was determined by flow cytometry after 12 h. The collected supernatants were added to fresh AK31 and CNE-2 cells to determine the infection rate after 12 h. (E) AK31 or Raji cells were co-cultured with in-cell infected iCNE-2 cells for 24 h. The expression of GFP in AK31 (upper) or Raji (lower) cells was analyzed by flow cytometry. AK31 and Raji cells only (left) served as negative controls.
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Related In: Results  -  Collection

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fig3: EBV from in-cell infected CNE-2 cells possesses enhanced infection potency and altered tropism. (A) Determination of GFP+ AK31 cells (left) and CNE-2 cells (right) after the infection with A-EBV from GFP-Akata cells (blue line) or C-EBV from iCNE-2 cells (red line) for 24 h by flow cytometry. Cells without EBV treatment served as negative control (black line). (B) Statistical comparison of percentages of CNE-2, A431, MCF-7, PLC/PRF/5, AK31 and NEPC1-Bmi1 cells with GFP expression after 24 h cell-free infection with A-EBV or C-EBV by immunofluorescence microscopy. All experiments were repeated at least three times. *P < 0.05. (C, D) Absorption assay of A-EBV (C) or C-EBV (D) was performed. 2 × 107 DNA copies/ml A-EBV (C) or C-EBV (D) virus was incubated with 2 × 106 AK31 or CNE-2 cells for 2 h on ice. Supernatants were collected and the fresh medium was added to cells for culture. The percentage of GFP+ AK31 or CNE-2 cells was determined by flow cytometry after 12 h. The collected supernatants were added to fresh AK31 and CNE-2 cells to determine the infection rate after 12 h. (E) AK31 or Raji cells were co-cultured with in-cell infected iCNE-2 cells for 24 h. The expression of GFP in AK31 (upper) or Raji (lower) cells was analyzed by flow cytometry. AK31 and Raji cells only (left) served as negative controls.
Mentions: We stimulated GFP-Akata and iCNE-2 cells with anti-human IgG49 and sodium butyrate52, respectively, to initiate the EBV virion production. After stimulation for 3 days, culture supernatants containing EBV virions were collected. To distinguish EBV virions derived from these two types of cells, A-EBV and C-EBV were named for EBVs from GFP-Akata and iCNE-2 cells, respectively. By using the same amount of virions normalized by quantitative PCR, we first performed the cell-free infection assay in susceptible AK31 and non-susceptible CNE-2 cells to compare the infectivity between A-EBV and C-EBV. A-EBV maintained the capacity to infect AK31 cells at the percentage of ∼10% but could not infect CNE-2 cells (Figure 3A left and right, blue line for A-EBV infection). Surprisingly, based on the expression of GFP fluorescence, we found that C-EBV from iCNE-2 cells infected both AK31 (Figure 3A, left, red line, 10.1%) and CNE-2 cells (Figure 3A, right, red line, 14.8%). It is well established that deficiency of EBV receptor such as CD21 molecules on ECs is the main obstacle for their susceptibility to cell-free EBV infection19. However, the extra ability of C-EBV to infect CNE-2 cells suggests that CNE-2 cells infected via in-cell infection are able to assemble virions with an extra capacity to infect non-susceptible ECs. This was confirmed when we further compared the capacity of A-EBV and C-EBV to infect other lymphocytes or EC-derived cell lines (Figure 3B). While A-EBV only infected B cell line AK31, C-EBV infected both lymphocytes (AK31) and EC cell lines (i.e., CNE-2, A431, MCF-7, PLC/PRF/5 and an immortalized epithelial cell line NPEC1-Bmi1)53.

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