A Cell-Based Examination of Modulators of Pre-Membrane Processing As a Target against Dengue Virus

Dengue Virus (DenV) is an arbovirus that represents a budding risk in the world. Every year, up to 100 million DenV infections manifest into Dengue Fever or in extreme cases Dengue Hemorrhagic Fever and Dengue Shock Syndrome. DenV research in vaccine development has proven to be a difficult feat due to the phenomenon of antibody dependent enhancement. Furthermore, there are currently no available antivirals to fight infection, viral protein processing or viral production. DenV lifecycle begins with its genomic release in the cytoplasm, where it is then translated as a single polypeptide embedded in the Endoplasmic Reticulum (ER) membrane. DenV, like so many other viruses, exploits a range of host enzymes in the Classical Secretory Pathway (CSP) for modifications. Among these important host enzymes are proteases such as the family of Proprotein Convertases (PCs), including furin. The modulation of the pre- membrane (pr-M) protein, most likely by PCs, is a critical step in the DenV lifecycle as an absence results in noninfectious progeny. Interestingly, the maturation of pr-M by the host enzymes during infection has been characterized as incomplete, thus rendering some viral particles noninfectious. Thus, the inhibition of pr-M cleavage presents an attractive target for potential antivirals. This assay is based on a fusion that contains an ER targeting signal sequence, the substrate of significance with flanking FLAG and HA epitopes, and a transmembrane (TM) domain. The assay in the context of the pr-M boundary has shown robust transportation with a wild phenotype in both transient and stable cell expression using retroviral technology. The original pr-M substrate included only 20 aa of the substrate boundary. Here, I have designed different substrate boundaries of pr-M to monitor important motifs in enzyme recognition and secretion, hypothesizing that by adapting to larger segments, we will have a powerful platform for the discovery of competitive inhibitors rather than inhibitors of the enzyme