Curnew, Leah J. F. (2023) Visualizing hepatitis C virus (HCV) core protein using an unnatural amino acid system. Masters thesis, Memorial University of Newfoundland.
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Abstract
Understanding the functionality and cellular location of viral proteins is key to unravelling the complexity of viral pathogenesis. Visualizing these proteins using microscopy is key to interpreting viral pathogenesis. Typically, antibodies and other traditional labelling mechanisms (GFP, tags, etc.) are used in microscopy to accomplish visualization. This is difficult to achieve when there are viral proteins for which no labelling mechanisms are available or when traditional labelling mechanisms produce artefactual results. The objective of this study was to visualize the hepatitis C virus (HCV) core protein using a non-traditional labelling mechanism to overcome instances when traditional labelling mechanisms are unavailable. We used the autofluorescent unnatural amino acid (UAA) 3-[(6-acetyl-2-naphthalenyl)amino]-L-alanine (ANAP) to visualize core via immunofluorescence and confocal laser scanning microscopy. Microscopic analyses showed successful incorporation of ANAP into core forming ANAP-containing core, which was found in association with cellular lipid droplets. These results suggest that core tolerates ANAP incorporation and ANAP incorporation does not disrupt core localization in cells, revealing that the UAA system can successfully label viral proteins. Here, we show that the UAA system can be used to visualize viral proteins without requiring antibodies or tags, and can potentially be used to label viruses for live cell and animal studies.
| Item Type: | Thesis (Masters) |
|---|---|
| URI: | http://research.library.mun.ca/id/eprint/16315 |
| Item ID: | 16315 |
| Additional Information: | Includes bibliographical references (pages 74-94) -- Restricted until January 1, 2025 |
| Keywords: | unnatural amino acids, HCV, HCV core, ANAP, immunofluorescence microscopy, confocal laser scanning microscopy |
| Department(s): | Medicine, Faculty of > Biomedical Sciences |
| Date: | October 2023 |
| Date Type: | Submission |
| Digital Object Identifier (DOI): | https://doi.org/10.48336/GXX8-3E97 |
| Medical Subject Heading: | Viral Proteins--metabolism; Hepatitis C; Amino Acids; Microscopy--methods; Fluorescent Antibody Technique; Antibodies |
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