Absorbance and Fluorescence Detection HPLC System and Its Projected Effectiveness in Analyzing Probed HIV-1 Proteins

1. Absorbance and Fluorescence Detection HPLC System and Its Projected Effectiveness in Analyzing Probed HIV-1 Proteins A Theoretical Project by Sarah L. Wood Faculty Advisor: Dr. Kenneth Overway

2. A review of the retrovirus HIV and how it infects a cell. http://health.howstuffworks.com/aids4.htm1

3. HIV on a CD4+ white blood cell Computer generated image Image from an electron microscope Pictures from http://www.avert.org/virus.htm

4. Human Immunodeficiency Virus Type 1 (HIV-1) • More virulent than HIV-2 2 • More likely to become AIDS 2 • Contains accessory proteins such as Vpr • Understanding these essential in AIDS prevention

5. Vpr, a virion-associated protein • Helps HIV propagation in several ways • Controls apoptosis 3 Left: untreated immune cells Right: treated with Vpr Picture from http://www.sciencenews.org/pages/sn_arc97/11_29_97/bob1.htm 4

6. Vpr, a virion associated protein • Increases transcription of the long terminal repeat • Same sequence of code at each end of a string of RNA or DNA 5 • Sticky ends used to insert HIV genome into host 5 • Contain protein binding sites involved in RNA initiation 5 Illustration from http://www.science-projects.com/v-DNA.htm

7. Other functions of Vpr in HIV-1 propagation • Helps HIV-1 preintegration complex containing viral DNA enter host cell • Cell cycle arrest in G2 phase

8. Benefits of fluorescence detection of proteins and metabolites • Easily tagged with fluorescent probes • Avoids purifying, tagging, and introducing labeled proteins into cells producing specific antibodies for cell’s antigens • Cell can be transfected with a cocktail of fluorescent protein variants for simultaneous study of multiple proteins

9. Probes useful for detection of HIV-16 • Tetramethylrhodamine-transferrin (TAMRA-TF) • Rhodamine red concanavalin (ConA) • Tetramethylrhodamine-5-malemide • LysoTracker Red • YOYO • BOBO • DiD • Green fluorescent protein (GFP)

10. Use of fluorescent proteins in visualizing HIV-1 Fluorescence microscope images of Hos/CD4 cells containing rhodamine-labeled tubulin (blue), membrane-denuded GFP-Vpr-labeled HIV (green) and membrane-encapsulated DiD-labeled HIV (red). Picture from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=533120 7

11. My instrument • Components • Quaternary high pressure gradient HPLC pump • Autosampler • Nucleosil column • Post-column derivatization system • Xenon lamp • Two monochrometers • Z flow cell • Five converging lenses • Five focusing lenses • Two photodiode arrays

12. Right half of diagram taken from http://www.towson.edu/~topping/exp8.pdf My Absorbance and Fluorescence Detection HPLC System With Gradient Elution Xenon lamp Converging and focusing lenses Post column derivatization system Photodiode array at 90o grating Z-flow cell lenses Reagent pump lenses waste grating Pressurized reagent reservoir Photodiode array for absorbance

13. Putting it all together • Infected cell must be lysed by HIV-1 specific cytotoxic T lymphocytes (CTL) • Autosampler injects a sample of the proteins • Use isocratic elution and allow solvent and sample to pass through the column • Column separates based on hydrophobicity • Proteins elute consecutively and react with the specialized fluorescent probes in the PCD system • Sample proceeds through flow cell • Detected by photodiode arrays • Sample exits to waste

14. Information given by my instrument • Basic chromatogram of intensity vs. time • Shows hydrophobicity of proteins • Shows relative abundance of proteins • Useful as HIV-1 fingerprint

15. Information given by my instrument • Fluorescence detector • chromatogram: instensity vs. wavelength • shows protein composition

16. Information given by my instrument • Absorbance Detector • Gives chromatogram with intensity vs. wavelength • shows spectral information concerning absorbance

17. Information given by my instrument • Three-dimensional chromatogram • x-axis = time • y-axis = absorbance or fluorescence • z-axis = wavelength

18. Three-dimensional chromatogram Absorbance or Fluorescence Protein A BOBO-1 Wavelength (nm) Protein B YOYO-1 TAMRA Lysotracker Red DiD Time (min)

19. Conclusions • Fluorescence detection of probed HIV-1 proteins seems to be a promising avenue for HIV-1 research advancement • Particular attention to Vpr may lead to medical breakthroughs in AIDS prevention • Instrument gives valuable information concerning HIV-1 such as fingerprint • Instrument may also be used for further biomedical studies such as DNA analysis or peptide separation

20. References • http://health.howstuffworks.com/aids4.htm • 2. Bugl, Paul. http://unhavax.hartford.edu/bugl/hiv.htm. October, 2001. • 3. Bourbigot, S., Beltz, H., Denis, J., Morellet, N., Roques, B. P., Mely, Yves, and Bouaziz, S. (2005) The C-terminal domain of the HIV-1 regulatory protein Vpr adopts an antiparallel dimeric structure in soluction via its leucine-zipper-like domain. Biochem. J., 387, 333-341. • 4. Fackelmann, Kathleen. http://www.sciencenews.org/pages/sn_arc97/11_29_97/bob1.htm. November, 1997. • Stowell, Dan. http://www.mcld.co.uk/hiv/?q=Long%20Terminal%20Repeat. • Stauber, Roland H., Rulong, Shen, Plam, Gottfriend, and Tarasova, Nadya I. (1999) Direct Visualization of the HIV-1 Entry: Mechanisms and Role of Cell Surface Receptors. Biochemical and Biophysical Research Communicaations 258, 695-702.

21. References • Watters, Christopher. (2004) Video Views and Reviews: Creating a Thread with Respect to the Invasion of Animal Viruses. Cell Biol Educ. 3(4), 218–222.