Review 1: "Inhibiting Glutamine Metabolism Blocks Coronavirus Replication in Mammalian Cells"

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:

The authors of this manuscript propose that coronaviruses use glutamine to metabolically fuel its replication. The inhibition of glutaminase reduces coronaviruses replication. 

The manuscript by Green et al aims at investigating the importance of Glutamine/Glutaminolysis for the replication of coronaviruses. The authors use different coronaviruses (Hcov-OC43, 299E and SARS-CoV) and different cell lines (HBEC, HCT8, MRC5 and VERO cells) to perform the experiments. Overall, the manuscript is well written and performed.

Overall, the work shows the need for glutamine for coronavirus replication. Important findings are:

1. Coronavirus infection changes the metabolic profile of the cell lines investigated

2. Coronaviruses induce de-expression of GLS

3. Knockdown of GLS reduces coronavirus RNA and Protein levels

4. GLS expression is modulated by JNK

5. JNK inhibition reduces coronavirus replication

6. Novel glutaminase inhibitors, SU1 and UP4, reduce coronavirus replication

However, there are some weaknesses that could improve the overall results: No experiments to evaluate the release of mature viral particles, as PFU assays, were performed; does supplementation with glutamine increase viral replication? What about glutamate or other TCA cycle intermediates? Cell viability after drug and sh/siRNA was not indicated.

The dependence of SARS-CoV-2 on glutamine has already been demonstrated under different circumstances, and would be interesting to be brought to discussion by the authors. Krishnan S et al. 2021 Mol Cell Proteomics, was among the first to reports robust changes in serum metabolomics of COVID-19 patients and the correlation with disease severity. The group highlighted a reduction of several aminoacids, including glutamine, arginine and tryptophan. Conversely, they showed for the first time that blockade of glutaminolysis with L-DON (6-Diazo-5-oxo-L-norleucine) reduced SARS-CoV-2 replication in Calu cells, although in a more simple way. Mullen PJ et al 2021 Nat Commun demonstrated by using U-13-Glutamine, to track the path of carbons derived from glutamine, important changes in glutamine metabolism and the fueling of TCA cycle during SARS-Cov-2 infection. Our group has confirmed these findings in astrocytes, where glutamine, lactate, piruvate and a-ketoglutarate were reduced, associated with changes in protein expression related to carbon metabolism. Conversely, the use of L-DON to block glutaminase, significantly reduced SARS-CoV-2 replication, whereas supplementation with glutamine increased the release of viral particles in PFU assays (Oliveira et al 2022 J Neurochem). Further, Krunfli F et al 2022 confirmed these results in human primary astrocytes. In summary, the research is relevant to the field, especially by indicating newer GLUS blocking drugs. However, adds few newer information on the metabolic changes caused by SARS-CoV-2 infection.