Reference no: EM132927526

The Non-structural Protein of Crimean-Congo Hemorrhagic Fever Virus Disrupts the Mitochondrial Membrane Potential and Induces Apoptosis.
Barnwal, B., Karlberg, H., Mirazimi, A., Tan, Y. (2016). THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 2, pp. 582-592, January 8, 2016

1) On which of the 3 RNA strands is the NSs gene sequence located?

2) Which one of the paper's authors was not listed as an author in the previously published data, reporting that CCHFV induces caspase 3 dependent apoptosis?

3) The authors report that they used an anti-actin monoclonal antibody obtained from Sigma. The Sigma website shows multiple different anti-actin monoclonals. What vital information have the authors om

4) Although it is not clearly stated in the Figure legend, 2 different types of antibody are used to detect native NSs in the CCHFV infected cells in Figures 2A and 2C.
i. What are the main steps in SDS-PAGE and Western blotting?
ii. Which antibodies did the authors use for the Western blots and the immunofluorescence experiments and why have they used different antibodies?

5) In Figure 2B the authors show the ratio of native or recombinant NSs to calnexin in cells infected with CCHFV or transfected with myc-tagged NSs.
i) Why was this deemed necessary, given that Western blots are shown in Figure
ii) How do the authors interpret the observation that at 48 hours post infection/transfection very little NSs is detectable in the absence of MG132, but in the presence of MG132, NSs is easily detectable?

iii) In the discussion the authors suggest that degradation of the NSs protein might be a host defence mechanism against viral infection, but why might suppression of NSs activity during the early stages of infection be advantageous to the virus.

6) What is the evidence that NSs can induce apoptosis in different cell lines and why did the authors decide to use the 293FT cell line for subsequent experiments?

7) In order to determine which amino acids might be important in the pro-apoptotic activity of NSs, the authors used an alanine scanning approach. What is alanine scanning mutagenesis?

8) Figure 5 presents the results of a series of experiments carried out to narrow down the key residues involved in NSs' pro-apoptotic activity.

Summarise the strategy used by the authors and interpret the evidence to cap3/7 activation presented in Figures 5A,C,E and G

9) In Figure 5A, the authors propose that full length NSs has reduced activity compared to truncated NSs carrying 48-140 and 93-140 and suggest that this might indicate the presence of an inhibitory sequence somewhere within the first 48 amino acids. Bearing in mind that NSs is subject to proteasomal degradation, suggest an experiment that could show if the first 48 amino acids were important in targeting NSs for degradation.

10) Putting yourself in the role of a reviewer, give an example of where you think the presentation of the Figures in the paper could have been improved and why.