Cellular Uptake of Modified Red Clover Necrotic Mosaic Virus and Small Molecule Release from the Virion

The ability to efficiently and specifically target cancerous cells with therapeutic agents would be a monumental advance in the area of cancer treatment. This research seeks to investigate the possibility of using a novel synthetic drug delivery vector to target human cervical cancer (HeLa) cells. This delivery vector is constructed by infusing small, positively-charged molecules into a plant virion?Red clover necrotic mosaic virus (RCNMV)?and then labeling the infused virion with multiple copies of a targeting peptide or agent. The infused molecules can be dyes for diagnostic applications or anti-cancer for chemotherapeutic applications. The targeting peptides can be of viral origin such as the nuclear localization sequence from the adenoviral fiber peptide. This peptide has the ability to target the CD46 receptor. The peptide can also be fluorescently labeled to monitor uptake in cells. The viral construct is delivered to HeLa cells, and cellular uptake of the conjugate and intracellular rhodamine release is simultaneously monitored. Once the virus is internalized, the decreased divalent calcium ion concentration and salt content of the cellular environment cause pores between coat proteins in the virion to open, and small, positively-charged molecules such as rhodamine can pass through these pores. Both the cellular uptake of the conjugate and rhodamine release are monitored using fluorescence activated cell sorting (FACS) flow cytometry. It has been found that degree of cellular uptake of the modified RCNMV constructs is a function of incubation time with the maximum uptake after a nine-hour incubation time. Furthermore, it has been determined that the amount of rhodamine released from the virion inside the cell increases with incubation time, but this release time lags behind the uptake time. Again, the maximum amount of rhodamine release is seen after a nine-hour incubation time. Once this system is fully characterized, studies of in vivo virus localization as well as encapsulation of probe molecules for detection of genes or proteins inside cells can be completed for diagnostic and research applications.