Visualizing HCV Core Protein via Fluorescent Unnatural Amino Acid Incorporation
<b>I</b><b>ntroduction</b><b>:</b> Unnatural amino acids (UAAs) share the same basic structure as proteinogenic amino acids. However, UAAs permit additional functions and applications to proteins due to their different side chains. Recent UAA applications include...
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MDPI AG
2020-07-01
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| Series: | Proceedings |
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| Online Access: | https://www.mdpi.com/2504-3900/50/1/129 |
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| author | Leah Jane Fitzgerald Curnew Kate McNicholas Bridgette Green Jackie Barry Hannah L Wallace Lingyan Wang Cassandra Davidson John P Pezacki Rodney S Russell |
| author_facet | Leah Jane Fitzgerald Curnew Kate McNicholas Bridgette Green Jackie Barry Hannah L Wallace Lingyan Wang Cassandra Davidson John P Pezacki Rodney S Russell |
| author_sort | Leah Jane Fitzgerald Curnew |
| collection | DOAJ |
| description | <b>I</b><b>ntroduction</b><b>:</b> Unnatural amino acids (UAAs) share the same basic structure as proteinogenic amino acids. However, UAAs permit additional functions and applications to proteins due to their different side chains. Recent UAA applications include using fluorescent UAAs to label proteins. The UAA system provides an alternative method to traditional protein labeling mechanisms (antibodies, GFP, and tags, such as HA and HIS), which can affect protein functionality and topology. The purpose of this study was to visualize the hepatitis C virus (HCV) core protein using the fluorescent UAA Anap (3-[(6-acetyl-2-naphthalenyl)amino]-L-alanine). <b>M</b><b>ethods</b><b>:</b> Huh-7.5 cells were co-transfected with HCV core plasmids containing amber stop codons at various positions throughout the coding sequence and a second plasmid encoding the orthogonal tRNA/synthetase pair that facilitates Anap incorporation. Three days post transfection, cells were stained for core protein and lipid droplets (LDs) and visualized using immunofluorescence or confocal microscopy. <b>R</b><b>esults</b><b>:</b> We have optimized transfection protocols for the efficient expression of the tRNA/synthetase pair required for Anap incorporation and are able to visualize our core mutant proteins containing Anap. We have successfully substituted Anap into 11 different positions within the core, including substitutions for tryptophan, tyrosine, and phenylalanine residues. In addition, we have shown that our core mutants associate with cellular LDs, suggesting that the incorporation of the UAA did not disrupt core protein expression, stability, or cellular localization. <b>C</b><b>onclusions</b><b>: </b>We have demonstrated the establishment of a UAA incorporation system in an HCV protein without any obvious impact on core protein function. The ability to label viral proteins using fluorescent UAAs eliminates the requirement of antibodies or tags for protein visualization. In conclusion, the UAA system is a useful method to study HCV proteins and can potentially be used to label viruses for live cell and animal studies. |
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| institution | Directory Open Access Journal |
| issn | 2504-3900 |
| language | English |
| last_indexed | 2024-03-10T18:30:28Z |
| publishDate | 2020-07-01 |
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| spelling | doaj.art-56bc7ca256e04be9867d06727b41bd0b2023-11-20T06:40:50ZengMDPI AGProceedings2504-39002020-07-0150112910.3390/proceedings2020050129Visualizing HCV Core Protein via Fluorescent Unnatural Amino Acid IncorporationLeah Jane Fitzgerald Curnew0Kate McNicholas1Bridgette Green2Jackie Barry3Hannah L Wallace4Lingyan Wang5Cassandra Davidson6John P Pezacki7Rodney S Russell8Memorial University, Immunology and Infectious Diseases, Division of Biomedical Sciences, Faculty of Medicine, St. John’s, NL A1B 3V6, CanadaMemorial University, Immunology and Infectious Diseases, Division of Biomedical Sciences, Faculty of Medicine, St. John’s, NL A1B 3V6, CanadaMemorial University, Immunology and Infectious Diseases, Division of Biomedical Sciences, Faculty of Medicine, St. John’s, NL A1B 3V6, CanadaMemorial University, Immunology and Infectious Diseases, Division of Biomedical Sciences, Faculty of Medicine, St. John’s, NL A1B 3V6, CanadaMemorial University, Immunology and Infectious Diseases, Division of Biomedical Sciences, Faculty of Medicine, St. John’s, NL A1B 3V6, CanadaMemorial University, Immunology and Infectious Diseases, Division of Biomedical Sciences, Faculty of Medicine, St. John’s, NL A1B 3V6, CanadaMemorial University, Immunology and Infectious Diseases, Division of Biomedical Sciences, Faculty of Medicine, St. John’s, NL A1B 3V6, CanadaDepartment of Biochemistry, Microbiology and Immunology and Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, ON K1N 6N5, CanadaMemorial University, Immunology and Infectious Diseases, Division of Biomedical Sciences, Faculty of Medicine, St. John’s, NL A1B 3V6, Canada<b>I</b><b>ntroduction</b><b>:</b> Unnatural amino acids (UAAs) share the same basic structure as proteinogenic amino acids. However, UAAs permit additional functions and applications to proteins due to their different side chains. Recent UAA applications include using fluorescent UAAs to label proteins. The UAA system provides an alternative method to traditional protein labeling mechanisms (antibodies, GFP, and tags, such as HA and HIS), which can affect protein functionality and topology. The purpose of this study was to visualize the hepatitis C virus (HCV) core protein using the fluorescent UAA Anap (3-[(6-acetyl-2-naphthalenyl)amino]-L-alanine). <b>M</b><b>ethods</b><b>:</b> Huh-7.5 cells were co-transfected with HCV core plasmids containing amber stop codons at various positions throughout the coding sequence and a second plasmid encoding the orthogonal tRNA/synthetase pair that facilitates Anap incorporation. Three days post transfection, cells were stained for core protein and lipid droplets (LDs) and visualized using immunofluorescence or confocal microscopy. <b>R</b><b>esults</b><b>:</b> We have optimized transfection protocols for the efficient expression of the tRNA/synthetase pair required for Anap incorporation and are able to visualize our core mutant proteins containing Anap. We have successfully substituted Anap into 11 different positions within the core, including substitutions for tryptophan, tyrosine, and phenylalanine residues. In addition, we have shown that our core mutants associate with cellular LDs, suggesting that the incorporation of the UAA did not disrupt core protein expression, stability, or cellular localization. <b>C</b><b>onclusions</b><b>: </b>We have demonstrated the establishment of a UAA incorporation system in an HCV protein without any obvious impact on core protein function. The ability to label viral proteins using fluorescent UAAs eliminates the requirement of antibodies or tags for protein visualization. In conclusion, the UAA system is a useful method to study HCV proteins and can potentially be used to label viruses for live cell and animal studies.https://www.mdpi.com/2504-3900/50/1/129unnatural amino acidsHCVHCV core proteinAnapimmunofluorescence microscopyconfocal microscopy |
| spellingShingle | Leah Jane Fitzgerald Curnew Kate McNicholas Bridgette Green Jackie Barry Hannah L Wallace Lingyan Wang Cassandra Davidson John P Pezacki Rodney S Russell Visualizing HCV Core Protein via Fluorescent Unnatural Amino Acid Incorporation Proceedings unnatural amino acids HCV HCV core protein Anap immunofluorescence microscopy confocal microscopy |
| title | Visualizing HCV Core Protein via Fluorescent Unnatural Amino Acid Incorporation |
| title_full | Visualizing HCV Core Protein via Fluorescent Unnatural Amino Acid Incorporation |
| title_fullStr | Visualizing HCV Core Protein via Fluorescent Unnatural Amino Acid Incorporation |
| title_full_unstemmed | Visualizing HCV Core Protein via Fluorescent Unnatural Amino Acid Incorporation |
| title_short | Visualizing HCV Core Protein via Fluorescent Unnatural Amino Acid Incorporation |
| title_sort | visualizing hcv core protein via fluorescent unnatural amino acid incorporation |
| topic | unnatural amino acids HCV HCV core protein Anap immunofluorescence microscopy confocal microscopy |
| url | https://www.mdpi.com/2504-3900/50/1/129 |
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