Localization of HA epitope tagged variants of bovine papillomavirus-1 E5 protein
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The bovine papillomavirus type 1 E5 protein is the smallest identified transforming protein. This protein alters expressed cellular phenotypes by causing activation of platelet-derived growth factor 𝛽 receptor (PDGF-𝛽 receptor) in the absence of ligand. During previous experiments, libraries of E5 variants with randomized transmembrane domains were assayed to test whether these altered proteins retained functionality as compared to the wild-type. One explanation for non-functionality of many of the clones posits that altered localization of the E5 variant protein leads to the inability of the variant to interact with the PDGF-𝛽 receptor. Because the localizations exhibited by proteins have been shown to be affected by their primary sequence, and natural signal sequences have been found to consist of short hydrophobic domains, I hypothesized that the sequence changes necessary for altered localization could be found entirely in the transmembrane domain of these small clones. Tests of HA and Au 1 epitope tags showed the former to exhibit less background staining. Six HA epitopetagged clones as well as an HA epitope-tagged wild-type E5 were then cloned into highexpression retroviral vectors and infected into C 127 murine cells. I then used indirect immunofluorescence to determine the subcellular localization of these variants as compared to the wild type. None of the tested clones exhibited the same localization pattern as the HA-tagged wild-type E5 protein, and one, of these clones showed striking differences. This supports the hypothesis that localization is affected by primary sequence and suggests that the localization of the clones could be linked to their functionality with further study.