RR:C19 Evidence Scale rating by reviewer:
Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.
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Review:
General Comments
In this manuscript evidence is presented that SARS-CoV-2 stimulates and interacts with lipid droplets (LDs) in infected human monocytes and different cell lines (A549, HMVEC-L, Vero E6). It is shown that in vitro infection of cells with SARS-CoV-2 reproduces the stimulation of LDs seen in ex vivo infected cells. Furthermore, chemical inhibition of a key enzyme of LD formation, diacylglycerol O-acyltransferase (DGAT-1), by compound A922500 inhibits the production of infectious progeny in SARS-CoV-2-infected cells. Modulation of pathways of lipogenesis in SARS-CoV-2-infected cells is observed. There is some co-localisation of viral proteins and the viral replicative dsRNA form with LDs. The pro-inflammatory innate immune response triggered by SARS-CoV-2 infection is reduced in A922500-pretreated, infected cells.
The data are of general interest, but deal with intracellular fatty acid biosynthesis and LD biogenesis in an incomplete way. The lack of uninfected, A922500-treated cells in Figs. 2 and 3 and their analyses is a major weakness. It may well be that the concentrations used of A922500 were non-toxic, but the evidence has to be presented in order to make the main data more convincing. The description of co-localisation data of viral proteins and viral replicative dsRNA with LDs should be rephrased
Specific Comments
Line
58 Consider citation of: Andersen KG, Rambaut A, Lipkin WI, Holmes EC, Garry RF. The proximal origin of SARS-CoV-2. Nat Med. 2020;26(4):450-452. doi:10.1038/s41591-020-0820-9
74 Consider citation of: Chatel-Chaix L, Bartenschlager R. Dengue virus- and hepatitis C virus-induced replication and assembly compartments: the enemy inside--caught in the web. J Virol. 2014;88(11):5907-5911.
78 Consider citation of: Cheung et al, 2010 (in ref list) and: Gaunt ER, Cheung W, Richards JE, Lever A, Desselberger U. Inhibition of rotavirus replication by downregulation of fatty acid synthesis. J Gen Virol. 2013a;94(Pt 6):1310-1317; Gaunt ER, Zhang Q, Cheung W, Wakelam MJO, Lever AML, Desselberger U. Lipidome analysis of rotavirus-infected cells confirms the close interaction of lipid droplets with viroplasms. J Gen Virol. 2013b;94(Pt 7):1576-1586.
85 Blockage of intracellular fatty acid (FA) biogenesis (and LD formation) by other compounds, such as TOFA, C75 and triacsin C, and its influence on viral replication have been described [Crawford SE, Desselberger U. Lipid droplets form complexes with viroplasms and are crucial for rotavirus replication. Curr Opin Virol. 2016;19:11-15; Lever A, Desselberger U. Rotavirus replication and the role of cellular lipid droplets: New therapeutic targets?. J Formos Med Assoc. 2016;115(6):389-394 (Table 1); Gaunt et al, 2013a, cited above]
96 For HCV consider citation of Lee et al, 2019 (in ref. list).
97 A reference relevant for noroviruses could be added: Doerflinger SY, Cortese M, Romero-Brey I, et al. Membrane alterations induced by nonstructural proteins of human norovirus. PLoS Pathog. 2017;13(10):e1006705.
102 … in vitro infection of cells and cell lines…
104 Suppl. Fig. S1. In panel A, a photograph of the uninfected cell A549 is missing.
126 A reason should be given why cells were treated with A922500 for only 2 hours before viral infection. The highest non-toxic and lowest effective doses of the compound have to be determined in the experimental system used.
128f and Fig. 2. In figure and text, relevant data on uninfected cells treated with A922500 are missing. The dose of 10 uM appears to be very high (with inhibitory doses 50% for different DGAT-1 enzymes being in the nanomolar range), and cells were pretreated with the compound for only 2 h. A dose response curve of treating uninfected cells with A922500 is missing. From experience with other inhibitors of the FA biosynthesis cascade it is known that inhibition of fatty acid biosynthesis and LD formation can be cytotoxic and that the chemotherapeutic index of inhibitory compounds is often low.
138f and Fig. 3. See comment on Fig. 2 above.
188 to line 199. Consider rephrasing, since SARS-CoV-2 antibody was used as a tool of detection of viral components. It is not clear which components were visualized, since a polyclonal convalescent serum was used. Figs. 4C and 4E show an incomplete merger of LDs and viral protein staining and viral dsRNA replicative form, respectively. Figs. 4C’ and 4E’ are not sharp and can be omitted.
233 … lipogenic phenotype… Please clarify and provide a reference.
251 Was it considered to infect cells with SARS-CoV-2 in the presence of oleic acid in the medium?
329 Fig. 1C uninfected is too dark.
333 Fig. 1E. The intracellular FA biosynthesis pathway is only partially shown. See Fig. 1 of Crawford and Desselberger, 2016 [cited above].
338 Fig. 1G. Consider phrasing: … Densitometric evaluation of data of panel 1F.
342 Fig. 2. Data on uninfected cells treated with compound A922500 are missing. See comment above.
354 Fig. 3. See comment to Fig. 2 (line 342).
386 Fig. S1. See comment above.
419 … endothelial cell line…
426f and 433f should be merged.
469 and line 474. Indicate the pH of buffers.
494 Consider citation of: Schmittgen, TD, Livak KJ. Analyzing Real-Time PCR Data
by the Comparative CT Method. Nat Prot 2008; 3 (6): 1101–8.
[827 words]
Minor Comments
Line
34 Consider reading: … biosynthetic needs. Thus, identifying…
38 … lipid metabolism, energy homeostasis and intracellular transport…
41 … were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARgamma, and DGAT-1 expression … and triggered LD formation in different human cell lines…
46 … mediators of pro-inflammatory response…
64 … obligate intracellular parasites… [Not all viruses are pathogens.]
68 … Increasing evidence points at… [see also line 200]
76 … enzymes… reduce viral replication…
85 … blocking LD biogenesis with a pharmacological inhibitor of … blocks viral replication… [Spell out DGAT-1 at first mentioning.
87 … uncover details of viral manipulation…
109 … insights into the mechanisms…
116 Omit ‘and remodeling’.
129 … inhibited the LD formation triggered by… infection in a dose-dependent way…
141 … changes in the homeostasis of cellular compounds…
182 … a high replicative capacity for SARS-CoV-2.
185 … the supernatant was used to measure the infectivity of progeny virus….
203 … To explore whether LDs are associated…
219 … participate in SARS-CoV-2 infection at two levels…
241 … Based on these data we suggest that these two transcription factors are important…
249 … that … infection increases… suggesting that the increase…
261 … is critical for LD biogenesis and mediates…
262 and line 269. … pharmacological suppression of ..
264 … can contribute to…
268 … decreased the viral load…
279 … SARS-CoV-2 infection … recapitulates…
283 … mediators of inflammation…
284 … inhibition of LD accumulation and…
558 Ref. Codo et al, 2020 is incomplete.
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