Peer review of: NSs amyloid formation is associated with the virulence of Rift Valley fever virus in mice (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The authors present a detailed study demonstrating that the virus-induced filament-like aggregation 
of its virulence factor (the protein NSs) in cells exhibits characteristics typical for amyloids, which are 
known to lead to human brain diseases. This is an interesting and important result. The authors use a 
whole palette of biochemical and microscopy tools to convincingly show the proof. The manuscript is 
very well written and structured, and the experiments well thought and performed. I would really like 
to see this nice story published in Nature Communications. 
 
I have a few comments though: 
- Lines 70ff: The authors should make clearer from the start here that this is what has been shown in 
this manuscript (and not in previous work) – I got confused. 
- General to STED and EM data: I would like to see comparing values on filament sizes also from the 
STED data at any time point and to compare it to the values from the EM data (should be resolvable in 
STED). In general, I would like to see more statistics on the values, maybe with specific plots (such as 
done in Figure 4, also add statistical significance values). 
-Line 182: Are these the HMWs identified in the western blots? This was unclear to me at first. 
- Line 213: Confocal or STED images? (Why only confocal (if the case)?) 
- Lines 219ff: Are these experiments on fixed or living cells? 
- Lines 229ff: How was fluorescence labelling done here? Live or fixed cells? 
- Line 239: I did not understand the meaning of the last part of this sentence? 
- Lines 245ff: Did the dye ReAsH produce any background that might introduce some additional 
biasing dynamics? I would like to see more statistics and values here instead of just one 
representative movie. 
- Lines 266ff: Fixed and immunolabelled (how) cells? 
- Lines 272ff: I would like to see an analysis/plots again like done for the previous measurements. 
How do the authors know that “these fibrils did not correspond to any of the known endogenous 
regular structures involving ….”? 
- Line 293: I would like to see a statistical analysis with values for the aggregate sizes here as well. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
NSs is the main virulence factor of RVFV that, although being intensively researched, acts by several 
mechanisms that are not really understood. Léger et al convincingly showed by a series of methods 
that the NSs is the first example of a viral protein that forms amyloid fibers. A relevance for 
pathogenesis is not clear, but very likely. 
 
Major points: 
- There are several inhibitors of amyloid formation that would be interesting to try on NSs. 
- From the effect of DTT on the resolution of NSs polymers it is concluded that NSs fibers are 
stabilized by disulfide bonds. However, the intracellular redox milieu is normally highly reducing. Upon 
cell lysis with buffers not containing reducing agents, oxidation happens quickly. So, strictly speaking, 
HMW smears on the SDS gel in the absence of DTT do not necessarily proof disulfide bond formation 
inside the cell. Of course virus infection means oxidative stress, so an increase in intracellular fibril 
growth may well be due to that. Experiments with antioxidants would help to solve this question. Thes 
results would also be important in the light of the discrepancies with the NSs structure paper by Barski 
et al., who did not find any disulfide bonds. 

 
Minor points: 
- The authors discuss the relevance of the fibrils for pathogenesis only in passing. Fibrils appear after 
4 ½ hours, but many anti-IFN activities of NSs take place earlier than that. A more profound 
discussion of the literature is necessary. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
Leger et al studied the localization and structure of the NSs virulence factor of RVFV in vivo and in 
vitro. They unexpectedly found by TEM, various fluorescence-based microscopy including amyloid-
specific dye and other techniques that NSs forms amyloid-like fibrillar structures in virally infected cell 
lines. Such amyloid fibrils are known to be disease-associated any may contribute to the virulence of 
RVFV. The fact that RVFV encodes an amyloidogenic protein is an important finding that offers 
interesting novel avenues for further research. However, there are some issues that dampen my 
overall enthusiasm of this fascinating finding. 
 
1. What is the role of the virally-encoded fibrils? Authors speculate a bit in the discussion but this is 
vague. There are examples in the literature that e.g. herpesviruses encode proteins that form 
amyloids with cellular proteins to inhibit necroptosis (Pham et al 2019, Embo). 
2. Authors state that RVFV is the first example of a virus that encodes amyloid. This is not correct (see 
paper above and McIntosh PB, J Virol. 2008 Aug;82(16):8196-203) 
3. Evidence for the presence of amyloid fibrils in tissue section obtained from the RVFV infected mice 
is missing. 
 
Minor issues: 
What happens with the NSs fibrils after virus-induced cell death of RVFV-infected cells? I.e., are fibrils 
found in the medium of infected cell cultures (or even in the blood/liquor of infected animals?)? 
Line 85 - What does “lacks most of the NSs” mean? Is something still being expressed? Is the virus 
hampered in infectivity? 
Fig 1 - Why was ThS not stained in the histological sections? The direct evidence for amyloids in vivo 
is missing. 
Line 91f/ Fig 1A/2A - As there is neither N or NSs visible in the staining of ΔNSs, is it possible that 
cells are not infected? In contrast to Fig. 1A, Fig. 2A shows N expression in the cytoplasm of U87 cells 
infected with the ΔNss virus. Why not in Fig. 1A? 
Line 105 - Is the formation of filamentous structures dependent on the MOI? 
Line 126/Fig 2 C-F - Why is there a switch from Vero cells (for STED) to HeLa cells (for TEM) back to 
Vero cells (for CLEM)? Why are different imaging methods used for each cell line tested? 
Line 140 - Similar to earlier point – why is one cell imaged with STED and another with TEM? Why not 
Vero for both methods? 
Line 143 - “None of the nuclear rosettes were found in cells infected with ΔNss virus” – please show 
this 
Fig S3A - Hard to distinguish between cell and fibrillar structure 
Line 150/Fig S3B - Why does this local annealing occur at such low frequencies? 
Line 163 - Binding of ThS does not unambiguously indicate amyloid fibrils, as the dye is specific for 
cross ß-structures 
Fig 3A - Why are there globular structures visible in the ThS signal for cells infected with ΔNSs strain? 
Line 186/Fig 3C - Is the fraction of NSs in the wells significant compared to the amount of protein in 
each lane? 
Line201 - How do disulfide bridges form in the reductive environment of the nucleus? 

Line233 - Fig 4 F and G Is there data available for in-between timepoints? 
Line 291 - As Vero cells are poorly transfectable, the fact that only 5% of cells expressed NSs might 
also just be because only those were transfected, not a specific effect of NSs.