In the lab, you are investigating a newly identified virus to determine how it i

Question

In the lab, you are investigating a newly identified virus to determine how it infects host cells. Luckily, you obtained a GFP - expressing virus from a colleague, so you can observe infection via fluorescent microscopy. You decide to use this tool to test whether clathrin-dependent endocytosis is the required entry mechanism for this virus.

A) Please describe a general experimental design to test this hypothesis through the use of genetic mutation, including the effect(s) of the protein mutation(s) you would introduce to the host cell.

B) Consider the anticipated results of this experiment. What are two results you may observe. What would each of these results suggest about how the virus enters the cell?

C) If you determined that the virus does require clathrin-dependent endocytosis. Name two other proteins that are most likely involved in the process of viral entry into the cell.

D) If you determined that the virus does not require clathrin-dependent endocytosis, what other mechanisms might be the way this virus enters cells?

Explanation / Answer

A. The gfp expressing virus when transducted into a particular cell line (say HEK 293) should colocalise with Clathrin protein or AP2 (adapter protein 2 complex). Genetic mutation of Clathrin proteins or Clathrin dependent proteins during viral entry due to which virus does not get endocytosis,rather they stay extracellular can help us to delineate the viral entry mechanism.

B. If the virus does not get endocytosed due to mutation but gets entry into the cell without mutation , the viral entry /endocytosis is clathrin mediated. Otherwise it may be faceplate dependent or other mechanism may explain viral entry.

C. Other proteins involved in clathrin mediated include adaptor protein complex 2(AP2) and EPs 15 protein

D. Other mechanisms may be caveolae dependent or macropinocytosis other than clathrin mediated for viral entry.

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