Formation of HERV-K and HERV-Fc1 Envelope Family Members is Suppressed on Transcriptional and Translational Level
Abstract
:1. Introduction
2. Results
2.1. ORFs of HERV-K18, HERV-K113, and HERV-Fc1 form Envelope Proteins
2.2. Analysis of Post-Translational Processing of HERV ENV
2.3. Analyzed HERV Envelope Proteins are not Transported to the Plasma Membrane but Reside within the Endoplasmic Reticulum
2.4. HERV Envelope Proteins Increase Gene Expression of Markers for Unfolded Protein Response but Trigger no Antiviral Response
2.5. Rare Leucine and Valine Codons Result in Decreased ENV Protein Synthesis in Human Cell Lines
2.6. Rare Codons have a Negative Effect on Expression of HERV-K113 Envelope Protein in a Cell-Free Expression System
2.7. Inefficient Synthesis of Native HERV ENV—A Problem of Secondary RNA Structure?
3. Discussion
4. Materials and Methods
4.1. Generation of Expression Plasmids
4.2. Cell Lines
4.3. Transfection
4.4. Protein Extraction
4.5. Deglycosylation
4.6. SDS-PAGE
4.7. Western Blot Analysis
4.8. Sample Preparation for Mass Spectrometry
4.9. LC-MS/MS Data Acquisition and Analysis
4.10. Flow Cytometry
4.11. Immunocytochemistry
4.12. RNA Extraction and cDNA Generation
4.13. Quantitative Real Time PCR (qRT-PCR)
4.14. Cell-Free In Vitro Transcription and Translation
4.15. In-Silico Determination of mRNA Secondary Structure
4.16. Statistics
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample | Protein | Sequence Coverage (%) | Proteolytic Peptides # |
---|---|---|---|
Control | K113, Fc1 envelope proteins | n.d. | n.d. |
ER chaperone BiP | 20 | 7 | |
K113 | K113 envelope protein | 4 | 3 |
ER chaperone BiP | 47 | 31 | |
coK113 | K113 envelope protein | 20 | 10 |
ER chaperone BiP | 48 | 27 | |
Fc1 | Fc1 envelope protein | n.d. | n.d. |
ER chaperone BiP | 53 | 36 | |
coFc1 | Fc1 envelope protein | 39 | 25 |
ER chaperone BiP | 54 | 38 |
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Gröger, V.; Wieland, L.; Naumann, M.; Meinecke, A.-C.; Meinhardt, B.; Rossner, S.; Ihling, C.; Emmer, A.; Staege, M.S.; Cynis, H. Formation of HERV-K and HERV-Fc1 Envelope Family Members is Suppressed on Transcriptional and Translational Level. Int. J. Mol. Sci. 2020, 21, 7855. https://doi.org/10.3390/ijms21217855
Gröger V, Wieland L, Naumann M, Meinecke A-C, Meinhardt B, Rossner S, Ihling C, Emmer A, Staege MS, Cynis H. Formation of HERV-K and HERV-Fc1 Envelope Family Members is Suppressed on Transcriptional and Translational Level. International Journal of Molecular Sciences. 2020; 21(21):7855. https://doi.org/10.3390/ijms21217855
Chicago/Turabian StyleGröger, Victoria, Lisa Wieland, Marcel Naumann, Ann-Christin Meinecke, Beate Meinhardt, Steffen Rossner, Christian Ihling, Alexander Emmer, Martin S. Staege, and Holger Cynis. 2020. "Formation of HERV-K and HERV-Fc1 Envelope Family Members is Suppressed on Transcriptional and Translational Level" International Journal of Molecular Sciences 21, no. 21: 7855. https://doi.org/10.3390/ijms21217855