1. What is a receptor and why do viruses interact with receptors? B) View the data in Figure 1 below and then answer Questions 2-7 below: Figure 1: Effect of an antimicrobial peptide (AMP) on virus infection. A) Sequence of the original antimicrobial peptide (AMP37) and two peptides in which specific amino acids were mutated to either glycine (GG-48) or A) AMP37 LLGDFFKPSILKWARMWINTERSWEATERPRTESSKL GG-48 WW-48 GILKWARMWINTERSWEATERGR WILKWARMWINTERSWEATERWR +Virus Mock No Peptide AMP37 GG-48 ww-48 000 00 0.01 μM 0.1 μM 1 μM 2. What is the objective of this experiment? 3. In this experiment, what is crystal violet staining? tryptophan (WW-48). Mutated amino acids are shown in red. B) Monkey kidney (Vero) cells were incubated without or with different concentrations of AMP37, GG-48 and WW- 48 for 2 hours before infection with virus. At the time of infection cells -/+ peptide were incubated withphosphate buffered saline (PBS; mock-infection) or with virus. After 1 hour, the virus and peptide were removed, and fresh media -/+ peptide was added onto the cells. 12 Hours later, the media was removed, the cells were fixed with 3.4% formaldehyde and the stained with crystal violet. Relative levels of viral RNA 4. Why is the well of the mock infected cells purple but there is no color in the well that was incubated with virus and no peptide?) 5. Rank the antimicrobial peptides in the order of potency. 6. Explain your ranking from Question 5. 7. What do you think these peptides are targeting (the virus or the cell)? Explain your answer. View the data in Figure 2 below and then answer Questions 8-11 below: Figure 2: Effect of an antimicrobial peptide (AMP) on virus infection. 500- 400- 300- 200- 100- O Mock GG-48 WW-48 *** *** T 6 8 10 12(h.p.i) Monkey kidney (Vero) cells were incubated without or with different GG-48 and WW-48 for 2 hours before infection withvirus. At the time of infection cells -/+ peptide were incubated with phosphate buffered saline (PBS; mock-infection) or with a single-stranded positive-sense RNA virus. After 1 hour, the virus and peptide were removed and fresh media -/+ peptide was added onto the cells. At 1-, 2-, 4-, 6-, 8-, 10- and 12-hours post- infection cells were collected and the amount of virus in the cells was quantified by RT-PCR that measured levels of viral RNA and cellular GAPDH mRNA. Statistical significance was determined by student t-test when comparing mock-infection versus treatment with the antimicrobial peptides. ***P<0.0001 8. Provide a brief description of the infectious cycle of a single-stranded positive sense RNA virus from the of entry into the cell to exist. 9. In Figure 2, how are the researchers measuring infection? 10. Describe the data presented in Figure 2 in detail. 11. At what step in the infectious cycle do you think the GG-48 and WW-49 peptides are acting to restrict virus infection? Explain your answer. Virus G protein 12. What is the mechanism used by enveloped viruses to transverse a cellular membrane and enter the cell? 13. What viral protein is used for this process? 14. Draw a schematic or describe the important features of this viral protein? View the data in Figure 3 below and then answer Questions 15-19 below: + Control plasmid + ADAM17 + Furin + Cathepsin K Figure 3: Vero cells were co-transfected with a plasmid expressing the glycoprotein (G) found on the surface of Lexo Virus (LexoV), an envelope virus, and different cellular enzymes (ADAM17, Furin and Cathepsin K). Membrane fusion was examined by the formation of syncytia or multinucleated cells 24 hours post-transfection. Arrows show syncytia. 15. Describe the data in Figure 3. 16. Does the LexoV glycoprotein require a cellular cofactor for membrane fusion? Explain your answer. 17. Where in the cell are ADAM17, furin and cathepsin K localized? 18. The LexoV G protein is synthesized as a precusor potein (G0) which is then proteolytically cleaved to G1 and G2. G1 interacts with the cellular receptor and G2 mediates membrane fusion. Knowing the subcellular location of ADAM17, furin and Cathepsin K, where in the cell is G0 likely proteolytically cleaved. 19. At which cellular membrane does the G2 protein promote membrane fusion and entry LexoV? Explain your answer. A) Use Figure 4 below to answer Questions 20-25: B) LexoV-PWT LexoV-P ΔΑΒ 4.5 5 5.5 6 6.5 7 11 4.5 5 5.5 6 6.5 7 h.p.t. 11 Band intensity (normalized to maximal intensity) 1.0- 0.5- → LexoV-PWT ...LexoV-PAAB Time post-transfection (h) Ţ LexoV is a single-stranded negative-sense RNA virus that encodes N, P, M, and L proteins. Use your knowledge of the VSV replication mechanisms to help answer the questions below. 20. Which viral proteins are required for replication of LexoV? Describe the function of each of these proteins. Figure 4: The goal of this experiment was to investigate the role of an unusual domain (AB) within the LexoV P protein. In this experiment, vero cells were infected with LexoV containing either the wildtype P protein (LexoV-Pwr) or a virus in which the AB domain within P was deleted. (LexoV-PAB). At 2 hours post-infection, the normal media was removed and replaced with phosphate-deficient media, [g-23P]ATP and actinomycinD. Cells were harvested every 30 minutes from 4.5-7 hours post-infection. Total cellular and viral RNA was isolated and separated in an agarose gel, dried and exposed to a phosphorimager screen to detect the amount of newly synthesized viral RNA. A) A representataive image of the viral RNA synthesized. B) Quantification of the viral RNA at each time point. * denotes the newly synthesized viral RNA. 21. Describe the overall strategy used by single-stranded negative-sense RNA viruses to replicate the viral genomes. 22. Describe the data shown in Figure 4A. 23. Describe the data shown in Figure 4B. 24. What conclusion can be drawn from Figure 4. 25. Speculate on the function of the AB domain within the P protein. Explain the rationale for your answer.