A Multiplexed Cell Based Platform For Monitoring Cleavage In Different Compartments
Roland Wolkowicz,
Associate Professor,
San Diego State University
Many human diseases are caused by RNA viruses including Human Immunodeficiency (HIV-1), Hepatitis C (HCV), Dengue (DenV) and more recently Chikungunya (CHIKV) or Zika (ZIKV) viruses. Robust cellular platforms are required to facilitate both phenotypic and targeted discovery. In order to provide a reliable and pinpointed approach for targeted discovery we utilize retroviral technology to engineer cell lines that monitor cleavage in relevant subcellular compartments. We previously developed an assay that monitors cleavage in a cytosolic environment for HIV-1 protease, which we are adapting to DenV, CHIKV and ZIKV proteases. We have more recently developed a platform that monitors the cleavage of the viral proteome in the classical secretory pathway, focusing on the premature membrane protein of DenV and WNV or the envelope of HIV-1. These are cleaved in the vesicles of the Golgi/TransGolgi Network (TGN. The assay relies on a two-tag system and cleavage events can be easily detected by flow cytometry or microscopy. Exploiting the power of fluorescent genetic barcoding we can multiplex the platform for different targets of the same virus or similar targets of different viruses. We have now adapted the platform to monitor cleavage at the cell surface or extracellular matrix (ECM), utilizing metalloproteinase 14 (MMP-14) as proof of principle. MMP-14 has an important role in the ECM restructuring, with important implications in cancer transformation and metastasis.
|
|
