Abstract
A recombinant measles virus (MV) which expresses enhanced green fluorescent protein (EGFP) has been rescued. This virus, MVeGFP, expresses the reporter gene from an additional transcription unit which is located prior to the gene encoding the measles virus nucleocapsid protein. The recombinant virus was used to infect human astrocytoma cells (GCCM). Immunocytochemistry (ICC) together with EGFP autofluorescence showed that EGFP is both an early and very sensitive indicator of cell infection. Cells that were EGFP-positive and ICC-negative were frequently observed. Confocal microscopy was used to indirectly visualize MV infection of GCCM cells and to subsequently follow cell-to-cell spread in real time. These astrocytoma cells have extended processes, which in many cases are intimately associated. The processes appear to have an important role in cell-to-cell spread, and MVeGFP was observed to utilize them in the infection of surrounding cells. Heterogeneity was seen in cell-to-cell spread in what was expected to be a homogeneous monolayer. In tissue culture, physical constraints govern the integrity of the syncytia which are formed upon extensive cell fusion. When around 50 cells were fused, the syncytia rapidly disintegrated and many of the infected cells detached. Residual adherent EGFP-positive cells were seen to either continue to be involved in the infection of surrounding cells or to remain EGFP positive but no longer participate in the transmission of MV infection to neighboring cells.
Overview
- The study aimed to rescue a recombinant measles virus (MV) that expresses enhanced green fluorescent protein (EGFP) and investigate its ability to infect human astrocytoma cells (GCCM).
- The methodology used for the experiment involved infecting GCCM cells with MVeGFP and using immunocytochemistry (ICC) and EGFP autofluorescence to visualize cell infection. Confocal microscopy was also used to visualize MV infection in real time and study cell-to-cell spread. The study used tissue culture to investigate the physical constraints governing the integrity of syncytia formed upon extensive cell fusion. The primary objective of the study was to understand the role of extended processes in cell-to-cell spread of MV infection in GCCM cells and the impact of physical constraints on the integrity of syncytia formed upon extensive cell fusion.
Comparative Analysis & Findings
- The study found that EGFP is an early and sensitive indicator of cell infection in GCCM cells. Cells that were EGFP-positive and ICC-negative were frequently observed, indicating that EGFP can be used as a reliable marker for cell infection. Confocal microscopy showed that MVeGFP utilized extended processes in GCCM cells to infect surrounding cells, and that heterogeneity was seen in cell-to-cell spread in what was expected to be a homogeneous monolayer. The study also found that physical constraints govern the integrity of the syncytia formed upon extensive cell fusion, and that residual adherent EGFP-positive cells may continue to be involved in the infection of surrounding cells or remain EGFP positive but no longer participate in the transmission of MV infection to neighboring cells.
Implications and Future Directions
- The study's findings suggest that EGFP can be used as a reliable marker for cell infection in GCCM cells and that extended processes play an important role in cell-to-cell spread of MV infection in these cells. The study also highlights the impact of physical constraints on the integrity of syncytia formed upon extensive cell fusion. Future research could explore the use of EGFP as a marker for cell infection in other cell types and investigate the role of extended processes in cell-to-cell spread of other viruses. The study also suggests that physical constraints may be important to consider when studying the spread of viruses in tissue culture and that future research could investigate ways to overcome these constraints.