The Biological Effect And Mechanism Of Cell-in-Cell Mediated Transmission Of Virus To Non-susceptible Cells

Cell-in-cell refers to the unusual structure with one or more viable cells existing inside another cell,which occur in most species ranging from lower organisms to mammals,and are known to be of biological significance.Cell-in-cell can occur in homogeneous cells(occurs most commonly between tumor cells)and heterogeneous cells(occurs most commonly between immune cells and epithelial cells or tumor cells).The fate of cells involved in cell-in-cell are diversified,but the death of the inner cell(term as effector cell)within outer cell(term as target cell)is the most common fate.Inflammatory and tumor tissues are the most common areas where cell-in-cells form in the human body,and the role of cell-in-cells in disease progression are becoming new hotspots in life sciences.During our in vivo cell-in-cell screening,we detected the prevalence of cell-in-cell structures formed by immune cells and epithelial cells(ECs)in tissues with microbial infection,such as in colon tissue from AIDS patients,Epstein-Barr virus(EBV)infected nasopharyngeal carcinoma tissues and tuberculous focus.We also found that in the EBV infected tissues,B lymphocytes and ECs in cell-in-cell structures were infected in varying degrees.Furthermore,in colon tissues from AIDS patients,HIV-1+ T lymphocytes could penetrate into ECs,some of which were also detected as HIV-1 p24 positive.Viral infection displays strict cell type specificity;EBV can infect both susceptible B lymphocytes and non-susceptible ECs,whereas HIV-1 is well known to specifically infect CD4+ immune cells,hence the existence of viral component in non-susceptible ECs suggests that viruses in target cells might have originated from the effector cells.Based on our in vivo tissue screening results and the “cell-in-cell stress model” proposed by our team,which indicates that outer target cells can acquire new biological features and modulate microenvironment after cell-in-cell structures were formed.We used Burkett's lymphoma(BL)-derived EBV-eGFP Akata cells which were infected by recombinant EBV with GFP reporter gene,and a typical EBV non-susceptible epithelial cell line CNE-2 to verify the implication of cell-in-cell in the transmission of EBV to ECs.We found that EBV-eGFP Akata cells could be engulfed by epithelial CNE-2 cells to form cell-in-cell structures in vitro.The formation of cell-in-cell structures led to the autonomous activation of EBV within EBV-eGFP Akata cells in an PI3K/AKT signaling pathway.The main fate of the inner cell is entotic death,however,some of the inner cells could live for a period of time or even escaped from CNE-2 cells.We also recorded the EBV infection of CNE-2 cells by time-lapse microscopy after cellin-cells were formed,as evidenced by the expression of GFP in CNE-2 cells.Significantly,EBV generated from infected CNE-2 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 NEPC1-Bmi1 cells,an immortalized nasopharyngeal epithelial cell line.Furthermore,we compared the cell-in-cell-mediated pathway with conventional cell-tocell-mediated pathway,through which we found that the transmission of EBV to ECs through cell-in-cell structures is independent of cell-to-cell contact or cell-free infection.The formation of cell-in-cell structures might be an alternative mechanism for viral transmission to nonsusceptible ECs,which we term as “in-cell infection”.We also used the HIV-1 infected T lymphoma cell line as the effector cells and uninfected ECs as the target cells to verify whether in-cell infection provides the medium for the transmission of HIV-1 to ECs.We observed that T lymphoma cells could efficiently penetrate into epithelial cancer cell lines,forming typical cell-in-cell structures.However,at the early stage of cell-in-cell formation,the T lymphoma cells undergo rapid apoptosis which was confirmed as emperitosis.By utilizing immunofluorescence staining and transmission electron microscopy analysis,we detected the existence of HIV-1p24 protein and HIV-1 like virions in the cytoplasm of ECs,which indicating that the outer cell could obtain the components of T lymphoma cell in a clean-up process after engulfment.We then packaged a recombinant HIV-1 that expressed GFP upstream of the viral early gene nef,which was used to infect the T lymphoma cells.It was observed after the formation of cell-in-cell structures that the outer ECs could not express GFP,which implies that the ECs were not effectively infected by HIV-1.We speculated that the rapid apoptosis at the early stage of cell-in-cell mediated the degradation of HIV-1 components,which delayed the timely assembly of the HIV-1 virions at the T lymphoma cell's membrane and influenced the subsequent virus transmission.In addition,different viruses may utilize different mechanisms in cell-in-cell mediated in-cell transmission process.Therefore,the interaction among different effector cells,viruses and their target cells in in-cell infection process may undergo different mechanisms and pathways.Chronic viral infection mediated by certain viruses such as EBV,HPV and HBV have been implicated in tumorigenesis,the persistent infection mechanism of those viruses have always been the focus of basic and clinical research.In this study,we first reported the prevalence of cell-in-cell structures in microbe infected tissues,and have 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 cell-in-cell not only participate in virus latency and replication,but exists as a general mechanism for EBV and other viruses to infect non-susceptible Ecs,the cell-in-cell structure formation thus participates in the pathogenesis of viral infection through its unique biological behavior.Further investigations on its molecular mechanisms will undoubtedly help us gain insight into viral transmission,and provide new clues and targets for the prevention and treatment of viral infections in clinic.