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

Cell-in-cell structures formed between B lymphocytes and nasopharyngeal ECs in NPC tissues. (A) Typical heterotypic cell-in-cell structures in one NPC tissue sample. Heterotypic cell-in-cell structures were indicated by yellow arrows. (B) Frequency of heterotypic cell-in-cell structures in NDNC (n = 3) and type 2b (III) NUNC (n = 22) determined by hematoxylin-eosin staining. The cell-in-cell frequency was scored with four scales: “−”, 0%; “+”, 1%-5%; “++”, 5%-10%; “+++”, 10%-15%. (C) Representative images of lymphocyte-nasopharyngeal EC cell-in-cell structures in a human NPC sample with co-staining of E-cadherin (green) and CD20 (red). DAPI staining (blue) indicated the nucleus. (D)EBER staining of NPC samples. Four types of heterotypic cell-in-cell structures were presented in the right lane. L−/E−: EBER− lymphocytes/EBER− ECs; L+/E−: EBER+ lymphocytes/EBER− ECs; L−/E+: EBER− lymphocytes/EBER+ ECs; L+/E+: EBER+ lymphocytes/EBER+ ECs. (E) Statistics of four types of heterotypic cell-in-cell structures in individual specimen indicated by different colors. (F) A representative TEM image of heterotypic cell-in-cell structure in tissue sections of NPC. In a typical cell-in-cell structure, the internalized B cell (indicated by a white arrow) was surrounded by the vacuole and the deformed nucleus (N) of the EC.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Cell-in-cell structures formed between B lymphocytes and nasopharyngeal ECs in NPC tissues. (A) Typical heterotypic cell-in-cell structures in one NPC tissue sample. Heterotypic cell-in-cell structures were indicated by yellow arrows. (B) Frequency of heterotypic cell-in-cell structures in NDNC (n = 3) and type 2b (III) NUNC (n = 22) determined by hematoxylin-eosin staining. The cell-in-cell frequency was scored with four scales: “−”, 0%; “+”, 1%-5%; “++”, 5%-10%; “+++”, 10%-15%. (C) Representative images of lymphocyte-nasopharyngeal EC cell-in-cell structures in a human NPC sample with co-staining of E-cadherin (green) and CD20 (red). DAPI staining (blue) indicated the nucleus. (D)EBER staining of NPC samples. Four types of heterotypic cell-in-cell structures were presented in the right lane. L−/E−: EBER− lymphocytes/EBER− ECs; L+/E−: EBER+ lymphocytes/EBER− ECs; L−/E+: EBER− lymphocytes/EBER+ ECs; L+/E+: EBER+ lymphocytes/EBER+ ECs. (E) Statistics of four types of heterotypic cell-in-cell structures in individual specimen indicated by different colors. (F) A representative TEM image of heterotypic cell-in-cell structure in tissue sections of NPC. In a typical cell-in-cell structure, the internalized B cell (indicated by a white arrow) was surrounded by the vacuole and the deformed nucleus (N) of the EC.

Mentions: To verify the presence of cell-in-cell structures in EBV-related NPCs, we first determined the existence of heterotypic cell-in-cell structures in NPC tissues. Indeed, we found that cell-in-cell structures were present in almost all clinical samples by hematoxylin-eosin staining (Figure 1A). The frequencies varied among subjects and NPC tissues (including nonkeratinizing differentiated nasopharyngeal carcinoma (NDNC) and nonkeratinizing undifferentiated nasopharyngeal carcinoma (NUNC); Figure 1B). Based on immunofluorescence staining, heterotypic cell-in-cell structures were characterized by the appearance of CD20+ B cells inside E-cadherin+ ECs (Figure 1C). Similar results were obtained with the determination of B cells by CD19 expression (data not shown). This was further confirmed by an independent study with samples from a different hospital (Wang S, data not shown).


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)

Cell-in-cell structures formed between B lymphocytes and nasopharyngeal ECs in NPC tissues. (A) Typical heterotypic cell-in-cell structures in one NPC tissue sample. Heterotypic cell-in-cell structures were indicated by yellow arrows. (B) Frequency of heterotypic cell-in-cell structures in NDNC (n = 3) and type 2b (III) NUNC (n = 22) determined by hematoxylin-eosin staining. The cell-in-cell frequency was scored with four scales: “−”, 0%; “+”, 1%-5%; “++”, 5%-10%; “+++”, 10%-15%. (C) Representative images of lymphocyte-nasopharyngeal EC cell-in-cell structures in a human NPC sample with co-staining of E-cadherin (green) and CD20 (red). DAPI staining (blue) indicated the nucleus. (D)EBER staining of NPC samples. Four types of heterotypic cell-in-cell structures were presented in the right lane. L−/E−: EBER− lymphocytes/EBER− ECs; L+/E−: EBER+ lymphocytes/EBER− ECs; L−/E+: EBER− lymphocytes/EBER+ ECs; L+/E+: EBER+ lymphocytes/EBER+ ECs. (E) Statistics of four types of heterotypic cell-in-cell structures in individual specimen indicated by different colors. (F) A representative TEM image of heterotypic cell-in-cell structure in tissue sections of NPC. In a typical cell-in-cell structure, the internalized B cell (indicated by a white arrow) was surrounded by the vacuole and the deformed nucleus (N) of the EC.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Cell-in-cell structures formed between B lymphocytes and nasopharyngeal ECs in NPC tissues. (A) Typical heterotypic cell-in-cell structures in one NPC tissue sample. Heterotypic cell-in-cell structures were indicated by yellow arrows. (B) Frequency of heterotypic cell-in-cell structures in NDNC (n = 3) and type 2b (III) NUNC (n = 22) determined by hematoxylin-eosin staining. The cell-in-cell frequency was scored with four scales: “−”, 0%; “+”, 1%-5%; “++”, 5%-10%; “+++”, 10%-15%. (C) Representative images of lymphocyte-nasopharyngeal EC cell-in-cell structures in a human NPC sample with co-staining of E-cadherin (green) and CD20 (red). DAPI staining (blue) indicated the nucleus. (D)EBER staining of NPC samples. Four types of heterotypic cell-in-cell structures were presented in the right lane. L−/E−: EBER− lymphocytes/EBER− ECs; L+/E−: EBER+ lymphocytes/EBER− ECs; L−/E+: EBER− lymphocytes/EBER+ ECs; L+/E+: EBER+ lymphocytes/EBER+ ECs. (E) Statistics of four types of heterotypic cell-in-cell structures in individual specimen indicated by different colors. (F) A representative TEM image of heterotypic cell-in-cell structure in tissue sections of NPC. In a typical cell-in-cell structure, the internalized B cell (indicated by a white arrow) was surrounded by the vacuole and the deformed nucleus (N) of the EC.
Mentions: To verify the presence of cell-in-cell structures in EBV-related NPCs, we first determined the existence of heterotypic cell-in-cell structures in NPC tissues. Indeed, we found that cell-in-cell structures were present in almost all clinical samples by hematoxylin-eosin staining (Figure 1A). The frequencies varied among subjects and NPC tissues (including nonkeratinizing differentiated nasopharyngeal carcinoma (NDNC) and nonkeratinizing undifferentiated nasopharyngeal carcinoma (NUNC); Figure 1B). Based on immunofluorescence staining, heterotypic cell-in-cell structures were characterized by the appearance of CD20+ B cells inside E-cadherin+ ECs (Figure 1C). Similar results were obtained with the determination of B cells by CD19 expression (data not shown). This was further confirmed by an independent study with samples from a different hospital (Wang S, data not shown).

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