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

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.

Implication of cell-in-cell structures in the transmission of HIV to epithelial cells

“…Thus, together with our work on EBV [2], we propose that the formation of cell-in-cell structures may have broader implications in spreading of different types of viruses. Future work based on this model would shed new light on how viruses evolve to adapt to new hosts and transmit between susceptible and non-susceptible cells.…”

“…ECs ( Figure 1C), suggesting that death of penetrated T cells may be involved in virus spread as seen in EBV incell infection [2].…”

“…Exosomes released from HIV-infected host cells have been reported to facilitate productive HIV infection in non-susceptible cells by delivering cargos such as virus proteins and RNA species [7]. Based on our previous discovery of EBV in-cell infection [2], we hypothesized that formation of heterotypic cell-in-cell structures could also lead to transmission of HIV from internalized CD4 + T cells to the non-susceptible ECs.…”

“…Recently, we reported that EBV could infect non-susceptible nasopharyngeal epithelial cells (ECs) through the formation of cell-in-cell structures between ECs and internalized B lymphocytes that have been infected with EBV. This novel mechanism was termed in-cell infection [2]. Given the formation of similar heterotypic cell-in-cell structures between other EC-lymphocyte pairs, such as epithelial tumor cells and T lymphocytes [3], we wonder whether in-cell infection also plays a role in mediating transmission of other viruses.…”

“…Future work based on this model would shed new light on how viruses evolve to adapt to new hosts and transmit between susceptible and non-susceptible cells. It should be noted that both cell-to-cell and cell-in-cell mechanisms could potentially contribute to EC infection in our cell co-culture system; actually cell-in-cell infection is more like an updated version of cell-to-cell infection as target and effector cells could make contact from head to toes within a limited space, thus leading to more efficient virus transmission [2]. While polarity has been implicated in HIV transmission via cell-to-cell [8] or exosome-mediated mechanisms [9], its role in in-cell infection of HIV or EBV remains to be investigated.…”